Detection of human hematopoietic stem cell engraftment in the livers of adult immunodeficient mice by an optimized flow cytometric method.

Abstract

Immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mice are a valuable resource to study human hematopoietic stem cells. Prolonged multilineage hematopoiesis indicates stem cell engraftment and generally is measured by flow cytometry. In this study, we took advantage of the multi-parameter detection afforded by modern flow cytometers to optimize detection of human hematopoiesis in NSG mice. Antigens widely expressed by mouse or human cells were evaluated as markers to distinguish mixtures of these cells to optimize and test the limits of chimerism detection. The bone marrow, spleen, and liver of NSG mice transplanted with human hematopoietic cells were analyzed for evidence of engraftment.Mouse bone marrow cells were best marked for exclusion by staining with a combination of CD45, TER-119, and anti-H-2K(d) monoclonal antibodies, whereas live human cells were most accurately identified by elimination of cell doublets and positive staining for CD59. Human stem cells (CD34(++)CD133(+)CD38(low)) and progenitors were detected in the bone marrow and liver, but not in the spleen. An unusual pattern of myeloid antigen expression was detected in the bone marrow and CD3(+)CD4(+)CD8(+) T-cells were detected in the spleen. We concluded that multicolor flow cytometric analysis that clearly distinguishes mouse and human cells offers accurate detection of human chimerism in NSG mice. Human hematopoiesis can be detected in the bone marrow and liver of NSG mice with T-lymphopoiesis, possibly occurring in the spleen.

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[Stem Cell Studies 2010; 1:e1][page 1]
Detection of human
an optimized flow cytometric
1
Francisco;
and Reproductive Sciences, University of
the Liver Center, University of California,
hematopoietic stem cell
method
engraftment in the livers of
Nicole L. Varga,1Alicia Bárcena,1,2
adult immunodeficient mice by
Marina E. Fomin,1 1Marcus O. Muench1,3
2 2Institute for Regeneration Medicine,
California, San Francisco;
San Francisco, California, USA
Abstract
study human hematopoietic stem cells.
Prolonged multilineage hematopoiesis indi-
cates stem cell engraftment and generally is
measured by flow cytometry. In this study, we
took advantage of the multi-parameter detec-
tion afforded by modern flow cytometers to
optimize detection of human hematopoiesis in
NSG mice. Antigens widely expressed by
mouse or human cells were evaluated as mark-
ers to distinguish mixtures of these cells to
optimize and test the limits of chimerism
detection. The bone marrow, spleen, and liver
of NSG mice transplanted with human
hematopoietic cells were analyzed for evidence
of engraftment.
Mouse bone marrow cells were best marked
for exclusion by staining with a combination of
CD45, TER-119, and anti-H-2Kdmonoclonal
antibodies, whereas live human cells were
most accurately identified by elimination of
cell doublets and positive staining for CD59.
Human stem cells (CD34++CD133+CD38low)
and progenitors were detected in the bone
marrow and liver, but not in the spleen. An
unusual pattern of myeloid antigen expression
was detected in the bone marrow and
CD3+CD4+CD8+T-cells were detected in the
spleen. We concluded that multicolor flow cyto-
metric analysis that clearly distinguishes
mouse and human cells offers accurate detec-
tion of human chimerism in NSG mice.
Human hematopoiesis can be detected in the
bone marrow and liver of NSG mice with T-
lymphopoiesis, possibly occurring in the
spleen.
1,2
1,3
1Blood Systems Research Institute, San
Department of Obstetrics, Gynecology,
3 3Department of Laboratory Medicine and
Immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/
SzJ (NSG) mice are a valuable resource to
Introduction
proliferation, and multilineage differentiation
into erythroid, myeloid, and lymphoid cells.
Although many markers have been identified
that help to isolate enriched populations of
hematopoietic stem cells such as CD34,
CD133, and lack of CD38 expression,1,2 human
stem cells cannot be isolated to absolute pur ity.
A number of in vitroculture systems have been
developed to measure the proliferative and
multilineage potential of hematopoietic pre-
cursors,3but none of these assays suffices to
measure stem cells among a mixed population
of precursors. Therefore, the ability of human
hematopoietic stem cells to reconstitute
hematopoiesis in vivo remains the gold stan-
dard for measuring functional hematopoietic
stem cells.4
Efforts are ongoing to develop more suitable
mouse strains that support human hemato -
poiesis and immune function.5Among the first
notable successes in this endeavor involved
the transplantation of human hematopoietic
tissues (liver and thymus or bone fragments)
into mice with severe combined immunodefi-
ciency, resulting in hematopoiesis mostly
localized to the site of the xenograft.6,7Use of
mice with multiple mutations, causing greater
immunodeficiency, allowed for seeding of the
murine bone marrow (BM) by intravenous
(i.v.) injection.8A combined approach using
fetal liver and thymus tissue grafts with i.v.
fetal liver infusion has been used to yield com-
plete hematolymphoid reconstitution in mice.9
NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are
among the most recently developed variations
of immunodeficient strains that support high
levels of reconstitution with human cells
owing to the complete lack of T-, B-, and NK-
cells in these animals.10These mice are excel-
lent recipients to study human hematopoiesis
and immune function.11
Flow cytometry has been an important tool
to evaluate human engraftment of mice. Early
studies were limited to analysis of only a few
simultaneous parameters owing to limitations
in the availability of fluorochrome-conjugated
monoclonal antibodies (mAbs) and the capa-
bilities of the most commonly available flow
cytometer. However, improvements in both
reagents and instrumentation offer the possi-
bility to advance the current means of detect-
ing human cells engrafted in immunodeficient
mice by staining samples with mAbs that dis-
tinguish murine and human cells. In concert,
this expands the characterization of the
engrafted cells to better understand the scope
of human hematopoietic reconstitution. We
probed the limitations of our current analytical
tools to detect human chimerism and investi-
Hematopoiesis is maintained by a pool of
stem cells capable of self-renewal, extensive
gated which antigens are the most effective
markers for evaluating multilineage reconsti-
tution in several hematopoietic tissues.
Materials and Methods
access, pathogen-free vivarium at the Blood
Systems Research Institute. Founder NSG
mice were obtained from Jackson Laboratories
(Sacramento, CA, USA). The described
research was performed with approval of the
Institutional Animal Care and Use Committee
Experimental mice
Mice were maintained in a restricted
Stem Cell Studies 2010; volume 1:e1
Correspondence: Marcus O. Muench, Blood
Systems Research Institute, 270 Masonic Ave.,
San Francisco, CA 94118, USA.
E-mail: mmuench@bloodsystems.org
Key words: hematopoietic stem cell, flow cytom -
etry, liver, chimeric mice, SCID mice.
Acknowledgements: the authors would like to
extend their appreciation and recognition to the
staff at the UCSF Medical Center for their contri-
butions to the research. We thank Dale
Hirschkorn for assistance with equipment, train-
ing, and management of the flow cytometry core.
We also thank the administrative staff at our
institute, in particular JoAnn Yates, Jerry
Michaelson, Abigail Schrock, and Barbara
Johnson. This work was supported by Blood
Systems Inc., and grants from the National
Institutes of Health: R21 HD055328, and support
through the University of California San
Francisco Liver Center (P30 DK026743).
Contributions: NLV acquired, analyzed, and
helped interpret data (Figures 1-3); AB partici-
pated in the conception and design of the study,
and helped in acquisition and interpretation of
data (Figures 5-10); MEF participated in the
acquisition of the data (Figures 1-10); MOM par-
ticipated in the conception and design of the
study, and contributed to the acquisition and
interpretation of data (Figures 1-10). NLV and
MOM contributed to drafting the manuscript, and
AB and MEF contributed to revising it. All authors
reviewed and approved the final manuscript.
Conflict of interests: the authors report no con-
flicts of interest.
Received for publication: 15 October 2010.
Revision received: 12 November 2010.
Accepted for publication: 12 November 2010.
This work is licensed under a Creative Commons
Attribution 3.0 License (by-nc 3.0).
©Copyright N.L. Varga, et al., 2010
Licensee PAGEPress, Italy
Stem Cell Studies 2010; 2:e1
doi:10.4081/scs.2010.e1
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[page 2][Stem Cell Studies 2010; 1:e1]
at ISIS Services LLC (San Carlos, CA, USA).
Mice were housed in sterile disposable
microisolator cages within laminar flow racks
(Innovive Inc., San Diego, CA, USA) and fed a
sterile, irradiated diet of Harlan Rodent Global
Diet 2919 (Harlan-Teklad, Madison, WI, USA)
with free access to sterile, irradiated water
(Innovive Inc.). Sterile irradiated corn-cob
bedding was used (Innovive Inc.) with auto-
claved cotton Nestlets (Ancare, Bellmore, NY,
USA), and GLP-certified Bio-Huts (Bio-Serv,
Frenchtown, NJ, USA) provided for environ-
mental enrichment. Entrance to the vivarium
was limited to trained authorized personnel,
and all husbandry and experimental proce-
dures were performed within a laminar-flow
cage-changing station (NuAire, Plymouth, MN,
USA).
Human hematopoietic cells
with approval of the University of California
San Francisco’s Committee on Human
Research. Gestational age of specimens
ranged from 18 to 24 weeks of amenorrhea;
age was estimated from the foot length of the
fetus. Human fetal bone marrow (FBM) was
attained as the primary source of hematopoi -
etic stem cells and progenitors for this study,
and was procured and isolated as previously
described.12Light-density fetal liver cells
depleted of mature blood cells were used as an
additional source of hematopoietic precursors
and were prepared as described.13
Fetal tissues were donated for research from
elective abortions with the consent of the
women undergoing the medical procedure and
Hematopoietic transplantation and
cGy using an RS2000 X-Ray Biological
Irradiator (RAD Source Technologies, Inc.,
Alpharetta, GA, USA) 1 to 3 hours before trans-
plantation. Mice were transplanted i.v. by tail
vein injection using a 28-gauge U100 insulin
syringe (BD, Franklin Lakes, NJ, USA) with
2¥107FBM cells or 1¥106lineage-depleted fetal
liver cells suspended in 200 μL of phosphate
buffered saline (PBS) (Mediatech, Inc.,
Manassas, VA, USA). After irradiation, the
standard rodent diet was replaced with irradi-
ated Global 2018 rodent diet with 4100 ppm
Uniprim®(Harlan-Teklad) for the first month
after radiation treatment.
Mice were sacrificed by carbon dioxide
asphyxiation followed by cervical dislocation at
ages ranging from 12 weeks to 1.5 years.
Human chimerism in NSG mice was analyzed
≥8 weeks after transplantation as indicated in
the text. BM was harvested by flushing the
femurs of the mice with approximately 1 mL of
culture medium using a syringe with a 27-
gauge needle (BD). Spleen and liver speci-
tissue harvest
NSG mice were irradiated with 175 or 200
mens were dissected and held in culture me -
dium until cell isolation. All tissues were held
on ice after harvest. NSG mice were examined
at the time of tissue extraction for visible signs
of poor health or physical abnormalities such
as tumors of the liver, spleen, and thymus. Any
moribund mice were removed from the study.
Hematopoietic cell and tissue pro-
7 minutes at 200X g at 4˚C and cells were
resuspended in blocking buffer for staining
with fluorochrome-conjugated mAbs. Blocking
buffer consisted of PBS supplemented with
0.01% NaN3(Sigma Chemical Co., St. Louis,
MO, USA), 5% normal mouse serum (Gemini-
Bio-Products, Inc., Woodland, CA, USA) and 2
μg/mL rat anti-mouse CD16/CD32 mAb
(BioLegend, San Diego, CA, USA).
Cell suspensions were prepared from mouse
spleens by passage through a 40 µm cell strain-
er (BD Biosciences, San Jose, CA, USA) in 1-2
mL PBS and layered on top of 1.077 g/mL
Lymphoprep (Axis-Shield PoC AS, Oslo,
Norway). Cells were centrifuged at 600X g for
30 minutes at room temperature and the light-
density fraction was collected. Collected cells
were washed with PBS and resuspended in
mouse blocking buffer.
Mouse liver cells were prepared by passage
through a 40 µm cell strainer and washed with
PBS by centrifugation (7 min at 200X g at 4˚C).
Cells were suspended in PBS, and light-dens ity
cells isolated, as for splenocytes and suspend-
ed in blocking buffer.
cessing
Mouse BM was washed by centrifugation for
Human bone marrow titrations
cell concentrations were first approximated
using a hemocytometer and trypan blue stain-
ing. A master mixture of approximately equal
numbers of pure mouse and human cells was
prepared and used for all further dilutions of
the human cells with NSG-BM. In a second
experiment, BM from a mouse engrafted with
human FBM was used as a master mixture of
cells. Triplicate or quadruplicate samples were
seeded in a 96-well plate and 1:2 serial dilu-
tions made with mouse BM. Cells were then
stained with anti-human and anti-mouse
mAbs as described. Samples of pure mouse and
human cells were used as controls. Because
hemocytometer cell counts have a low accura-
cy, we relied on the flow cytometric measure-
ments of the frequencies of human cells in the
master mixture and the degree of human cell
dilution to establish the calculated percentage
of human cells. Flow cytometry data typically
contained >105events/sample and are reported
as the mean ± standard error (SE) of individ-
To test the sensitivity of flow cytometry to
enumerate chimerism, human FBM was mixed
with BM from untransplanted NSG mice. Live
ual measurements. The significance of differ-
ences between samples was determined using
the unpaired t-test (Aable 3.0 software,
Gigawiz Ltd. Co., OK, USA), and differences
were considered significant at P≤0.05.
Flow cytometry
utes.13Table 1 lists the mAbs used in this study.
Conjugated non-specific antibodies used as neg-
ative controls were purchased from Invitrogen,
BD Biosciences and BioLegend. Samples were
washed twice with PBS supplemented with 0.3%
bovine serum albumin (Roche Diagnostic
Corporation, Indianapolis, IN) and 0.01% NaN3,
and suspended in the same solution containing
2 μg/mL propidium iodide (PI; Invitrogen,
Carlsbad, CA, USA). Samples were analyzed
using an LSR II flow cytometer (BD Biosciences,
San Jose, CA, USA). Data were analyzed using
FlowJo software, version 8.8 (Tree Star, Inc.,
Ashland, OR, USA).
Cells suspended in blocking buffer and held
on ice were stained with saturating levels of flu-
orochrome-labeled mAbs for at least 30 min-
Results
Our goal was to devise panels of mAb for the
analysis of human chimerism that dedicated a
single fluorochrome channel to staining all
murine BM cells and a single channel dedicat-
ed to detecting all human cells. Ideally, this
would allow for the greatest possible separ -
ation of murine and human cells and exclusion
of double-positive events that cannot be reli-
ably classified as human or murine in origin.
To allow the greatest flexibility in the choice of
mAbs and fluorochrome conjugates to evaluate
the human cells, the flow cytometry channels
selected for marking pan-murine and pan-
human cells should avoid using the most com-
monly applied fluorochromes such as fluores-
cein isothiocyanate (FITC), phycoerythrin
(PE), and allophycocyanin (APC).
Preliminary studies indicated that three
broadly expressed murine markers yielded
strong staining when conjugated to pacific blue
(PB): TER-119 (pan-erythrocyte), CD45 (pan-
leukocyte), and H-2Kd(pan-nucleated cells)
(Figure 1). Either CD45 or H-2Kdstained less
than half of BM cells because of the large num-
bers of erythrocytes present in the tissue.
Combining TER-119 and CD45 led to strong
staining of nearly all BM cells with a noticeably
higher mean fluorescence intensity (MFI) than
staining with TER-119 and H-2Kd. Because H-
2Kdcan stain non-hematopoietic cells present
in the BM, we tested a combination of all three
murine markers, which did yield the overall
Optimizing the detection of mouse
bone marrow cells
Article
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[Stem Cell Studies 2010; 1:e1][page 3]
highest and brightest staining.
Optimizing the detection of human
antigens found on a broad array of human
hematopoietic cells (Figure 2). Although
CD45, CD48, B2M, and anti-HLA-ABC could
stain the majority of leukocytes and/or nucleat-
ed cells, only CD59 (protectin) alone could
mark nearly all FBM cells. CD59 is expressed
by mature erythrocytes seen as cells with a low
forward light-scatter in Figure 2. Thus, we
used CD59 mAb conjugated with AF700 to iden-
tify total human cells. We have found an initial
screening of transplanted mice with a combi-
nation of CD34, CD45, CD59, and B2M to be
useful in identifying which mice show signs of
human reconstitution (Figure 2B). This panel
of pan-leukocyte markers is further useful in
identifying cases of low-level engraftment,
because detection of a small number of posi-
tive events using only a single marker can
result from non-specific staining of mouse BM
cells, whereas co-expression in the particular
pattern shown in Figure 2B indicates human
engraftment. It is worth mentioning that B2M
is a particularly bright marker of nucleated
human cells with the brightness of staining
using an FITC-conjugated mAb on par with
staining using PE-conjugated CD45. B2M may
be an ideal human marker of non-hematopoi-
etic cell types transplanted in mice. The addi-
tion of CD34 to the panel allows one to gauge
whether the presence of human cells is a
result of hematopoietic engraftment or simply
the presence of mature hematopoietic cells
such as erythrocytes (CD59+CD45–B2M–) or
leukocytes (CD59+CD45+B2M+).
cells
We evaluated a number of mAbs recognizing
Defining the limits of human cell
determine the sensitivity of flow cytometry in
detecting human cells among mouse BM cells
(Figure 3). Human CD59+cells were readily
detected in diluted mixtures of human FBM
and NSG-BM to levels below 1%. The sensitivi-
ty of detecting human cells with CD59, CD45,
or B2M was compared because staining with
the latter two markers is generally brighter,
offering greater differentiation from murine
cells, than staining using CD59. Although
CD45 and B2M staining could detect a signifi-
cant presence of human cells below 0.04%
(Figure 3C and D), these results were equiva-
lent to the sensitivity of CD59 staining (Figure
3B) in detecting the same level of FBM dilu-
tion. The weaker expression of CD59 was com-
pensated for by the greater number of human
events that could be detected, because CD59 is
also expressed on erythrocytes. The effective-
ness of using CD59 staining was confirmed
detection
We performed titration experiments to
further by measuring human cell engraftment
of NSG-BM harvested 223 days after transplan-
tation with FBM (Figure 3E). Significant
detection of human cells occurred at a calculat-
ed dilution of 0.32% human cells.
Doublet discrimination further
Chimeric mouse-human BM likely results in
aggregates of mouse and human cells that may
fail to be dispersed prior to analysis, leading to
events expressing both mouse and human
markers. We tested using doublet discrimina-
tion to remove cell clumps from analysis,14uti-
lizing BM from a chimeric animal with approx-
imately even numbers of mouse and human
cells (Figure 4). Plotting forward light-scatter
height versus area enabled nearly 3% of dou-
blet events to be eliminated from analysis,
improves the accuracy of human
cell detection
reducing the number of events stained with
both human and murine markers.
Detection of human hematopoietic
Hematopoietic stem cells are a rare popula-
tion of cells found among hematopoietic pre-
cursors expressing the highest levels of CD34
(CD34++),15expressing CD133,16and exhibit-
ing low/negligible levels of CD38 (CD38low).17,18
In mice with robust human engraftment, a
population of CD34++CD133+CD38lowcells was
readily detected in the BM (Figure 5). Even in
cases of low level engraftment, these cells can
be detected if a sufficient number of cells are
analyzed. In one case in which we analyzed
>1.2 million events, human engraftment was
detected at <0.2% and we observed a spectrum
of cells expressing
stem cell populations in the bone
marrow and liver
CD34, including
Article
Table 1. mAbs used in this study.
Antibody/Antigen Label
mAbs recognizing widely-expressed human antigens
FITC IgG1
AF488 IgG1
PEIgG1
FITCIgG1
PEIgM
AF700IgG2a
mAbs recognizing human progenitor and stem cell antigens
APCIgG1
PE/Cy7IgG1
PE IgG1
APCIgG1
mAb recognizing human erythroid antigens
APCIgG2b
mAbs recognizing human myeloid antigens
APCIgG1
PEIgM
APCIgG1
FITCIgM
mAbs recognizing human megakaryocyte and platelet antigens
FITCIgG1
PE IgG1
mAbs recognizing human B-cell antigens
APCIgG1
FITCIgG1
PE IgG1
mAbs recognizing human T- and NK-cell antigens
PE/Cy7IgG1
PE IgG1
APCIgG1
FITCIgG1
mAb recognizing mouse antigens
PB Rat IgG2b
PBMouse IgG2a
PBRat IgG2b
Isotype CloneManufacturer
b2-Microglobulin (B2M)
HLA-ABC
CD45
CD45
CD48
CD59
2M2
DX17
HI30
HI30
Tü145
p282(H19)
BioLegend
BD Biosciences
BioLegend
Invitrogen
BD Biosciences
BioLegend
CD34
CD34
CD38
CD133
581
581
HB7
AC133
BioLegend
BioLegend
BD Biosciences
Miltenyi Biotec Inc.
CD235aGA-R2 (HIR2)BD Biosciences
CD14
CD15
CD33
CD66b
HCD14
V1MC6
WM53
G10F5
BioLegend
Invitrogen
BioLegend
BioLegend
CD41a
CD42b
HIP8
HIP1
BD Biosciences
BioLegend
CD19
CD20
CD22
HIB19
L27
HIB22
BioLegend
BD Biosciences
BioLegend
CD3
CD4
CD8
CD56
UCHT1
L200
RPA-T8
HCD56
BioLegend
BD Biosciences
BD Biosciences
BioLegend
CD45
H-2Kd
TER-119
30-F11
SF1-1.1
TER-119
BioLegend
BioLegend
BioLegend
mAbs, mouse antibodies; FITC, fluorescein isothiocyanate; AF488 or AF700, alexa fluor 488 or 700; PE, phycoerythrin; APC , allophycocyanin;
Cy7, cyanine; PB, pacific blue.
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[page 4][Stem Cell Studies 2010; 1:e1]
CD34++CD133+CD38low
0.07% of human cells (data not shown). Such
findings indicate that enumeration of
hematopoietic stem cell populations is feasible
even in mice with very low levels of human
chimerism.
Interestingly, a typical phenotypic profile
reflecting active hematopoiesis was also seen
in the liver. A clearly defined population of
CD34++CD133+CD38lowcells was present as
well as cells expressing lower levels of CD34
and higher levels of CD38 characteristic of
committed hematopoietic progenitors. Similar
results were observed in numerous mice ana-
lyzed whenever human engraftment was also
detected in the BM. In contrast to the liver, very
few, if any, CD34+/++cells were observed in the
spleens of engrafted mice.
cells representing
Detection of human erythrocytes
precursors can also be used to help reliably
detect these cells by excluding large cells or
cells with a high side-light scatter from consid-
eration. Erythrocytes were detected based on
these criteria in the BM of a mouse transplant-
ed with FBM cells 229 days prior (Figure 6).
CD235a+cells were also observed among
splenic cells, suggesting circulation of human
erythrocytes. Note that light-density spleen
cells were analyzed, thus likely reducing the
number of mature erythrocytes detected
because of their high density. CD235a expres-
Erythrocytes are readily detected by their
specific antigen CD235a (glycophorin A).19The
relative small size of erythrocytes and their
Article
Figure 2. Comparison of cell-surface markers for the identification of human cells.
Human FBM cells were stained for a panel of cell-surface markers widely expressed by
hematopoietic cells (A). Note that only CD59 was expressed on nearly all events relative
to control staining (IgG1). FBM cells were stained with B2M-FITC, CD45-PE, CD34-
APC, and CD59-AF700 (B). CD34 expression is shown in blue in all three dot-plots.
Figure 1. Comparison of cell-surface markers for the identification of mouse cells. The expression of TER-119, CD45 and H-2Kdon BM
cells isolated from an untransplanted NSG mouse was examined. All mAbs were conjugated to PB. Numbers above the gates represent
the percentage of events falling within the gate. Additionally, the mean fluorescence intensity (MFI) is shown for cells stained with com-
binations of mAbs. An overlay of samples stained with TER119+CD45 (red line) and TER119+CD45+H-2Kd(blue line) is shown for a
second independent sample of NSG-BM indicating that the highest intensity staining is achieved with all three mAbs.
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[Stem Cell Studies 2010; 1:e1][page 5]
sion increases during erythrocyte maturation
and, accordingly, immature nucleated erythro-
cytes can be identified by their low level of
CD235a expression. Such cells were observed
in the BM, but to a lesser extent in the spleen.
Detection of human myeloid cells
by flow cytometry. We compared CD33 expres-
sion, a pan-myeloid cell antigen that has been
used to define myeloid reconstitution,19to
expression of CD66b, CD14, and CD15 (Figure
7A). These three antigens are less widely
Myelopoiesis generates a number of cell lin-
eages with distinct physical properties and cell
antigen expression that can be distinguished
expressed, with CD66b expression found on
neutrophils,20high CD14 expression represent-
ing cells of the monocyte lineage,21and CD15
expressed on myeloid precursors and mature
granulocytes.19Each of these three antigens
was expressed by a subset of CD33+cells recov-
ered from engrafted mice. The data also show
that a subpopulation with the highest CD33
expression is not co-stained with either
CD66b, CD14, or CD15, indicating that CD33
defines a larger population of human cells than
can be detected by a combination of the three
other antigens. Comparisons of CD66b, CD14,
and CD15 co-expression revealed several
popu lations of cells defined by these markers
in chimeric mice (Figure 7B) and fresh human
FBM (Figure 7C). In FBM, distinct CD14+
CD15–CD66b–monocytes and CD14–CD15+
CD66b+granulocyte populations were evident,
whereas in chimeric BM a similar pattern was
observed as well as a population of cells
expressing very high levels of CD66b and
CD15, with a high side-light scatter profile, but
lacking CD14 expression. There was an addi-
tional population of cells co-expressing CD66b
and variable levels of CD14 (Figure 7B) that
was not apparent in FBM. These data suggest
some differences in myeloid antigen expres-
sion in mice engrafted with FBM compared to
freshly harvested FBM.
We used the co-expression of two adhesion
molecules, CD41a22and CD42b,23to identify
mature megakaryocytes and platelets in the
BM and spleen of transplanted mice (Figure
8). CD41a+CD42b+events consistently repre-
sented a greater frequency of total human cells
among light-density spleen cells than among
total BM cells. CD41a+CD42b+events in the
spleen were represented by two distinctly sized
populations as viewed using forward light-
scatter (data not shown), presumably repre-
senting small platelets and larger megakaryo -
cytes. In the BM, the CD41a+CD42b+events
were more evenly dispersed within the light-
scatter gate used. CD41a+CD34+events were
also detected in the BM in animals with robust
reconstitution (data not shown). These pro-
genitors have been reported to be enriched in
megakaryocyte progenitors.24
Detection of human lymphoid cells
and by activated B-cells, was detected on
CD19+cells. CD22 (Siglec-2) is a B-cell marker
expressed on early progenitors and mature
cells, was also detected on CD19+and
CD19+CD34+cells, indicating that the full
range of human B-cell precursors can be
observed in the reconstituted mouse BM.
T-cells were sometimes detected in trans-
CD19 was used as the primary marker of the
B-cell lineage (Figure 9). CD20, which is
expressed in the latter stages of differentiation
Article
Figure 3. Sensitivity of flow cytomet-
ric detection of human fetal bone
marrow cells mixed with mouse bone
marrow. Human cells were analyzed
alone or mixed with NSG-BM cells
over a range of 0.006% to 100% cal-
culated based on the dilution of
human cells (A). Human cells were
defined as CD59-AF700+events that
were negative for staining with PI and
the murine mAbs TER-119-PB,
CD45-PB, and H-2Kd-PB. Note that
the data are presented on a logarith-
mic scale. A subset of these data for
dilutions containing <1% human
cells is shown in (B). Only in samples
containing a calculated 0.81% human
cells or higher was there a significant
(P≤0.05, asterisk) presence of human
cells detected compared to samples of
pure murine BM. A gray box indicates
1 SE above and below the mean of the
pure murine BM measurements
(n=3). The same samples shown in (B)
were also stained with CD45-PE and
B2M-FITC, for which the percentages
of positive events are shown in (C)
and (D), respectively. Detection of
human CD59+cells in BM harvested
from an NSG mouse transplanted
with human FBM diluted with vary-
ing amounts of untransplanted NSG-
BM is shown in (E). Data are shown
as the mean±SE, n=3-4 samples.
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[page 6][Stem Cell Studies 2010; 1:e1]
planted mice, but most often only after ≥3
months. Two examples of mice with full lymph -
oid reconstitution 229 days after FBM trans-
plantation are shown in Figure 10. T-cells were
defined by their small size using a typical
lymphoid-cell gate based on light-scatter prop-
erties and their expression of high levels of
CD3 and CD45. CD4 and CD8 expression
among these cells indicated that most of these
T-cells were single positive cells, although
some double positive events were observed. T-
cells, when present, were more frequently
observed among the human light-density
splenocyte population than in the BM.
Natural killer (NK)-cells, defined as
CD56+CD3–cells expressing high levels of
CD45 (Figure 10), were infrequently detected.
These cells were only observed in the BM of
mice that exhibited otherwise robust multilin-
eage human reconstitution. T-cells were more
often detected than NK-cells. We have
observed little or no expression of CD56 on
CD3+cells, thus most CD56+events are likely
to represent NK-cells. The expression of CD8
by some of the CD56+cells further indicates
that these cells are indeed NK-cells.
Discussion
channels offers the ability to sensitively detect
and better characterize human cells trans-
planted into immunodeficient mice. We
describe a seven-color staining protocol that
offers a high degree of specificity in detecting
human cells and flexibility in choosing labeled-
mAbs to analyze the engrafted cells. Total
human cells are detected by their expression of
CD59 and their lack of strong staining by three
markers of mouse cells: TER-119, CD45, and
anti-H-2Kd. Additionally, staining for cell viabil-
ity using PI and use of doublet discrimination
allows for accurate detection of human cells in
mouse BM at levels below 1%. Four fluores-
cence channels were used for lineage analysis
The capacity of modern flow cytometers to
analyze more than just a few fluorescence
Article
Figure 4. Improved detection of human cells in mouse bone marrow using doublet discrim-
ination. A live cell gate was used to eliminate dead cells stained with PI as shown (top-left).
Untransplanted NSG-BM (center column) and a mouse transplanted with human FBM
229 days prior (right column) were analyzed using only live cell gating (top row) or live cell
gating and doublet discrimination (center row). The staining pattern of doublet mouse
and/or human cells, excluded by the doublet discrimination, is shown in the bottom row.
Numbers represent the percentage of cells that fall within the gated regions.
Figure 5. Detection of human hematopoi -
etic precursors in mouse bone marrow and
liver. BM, liver, and spleen (rows top to bot-
tom, respectively) were examined from a
mouse transplanted 68-days prior with
human lineage-depleted fetal liver cells.
Hematopoietic precursors were stained
with CD34-PE-Cy7, CD38-PE, and
CD133-APC. CD133 staining is indicated
in red, highlighting a population of prob -
able hematopoietic stem cells in the BM
and liver with
CD133+CD38lowCD34++. All data were
gated for PI–CD59+events excluding mouse
cells as shown in Figure 4.
the phenotype
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[Stem Cell Studies 2010; 1:e1][page 7]
of the human cells in our staining protocol,
which is sufficient to perform a basic analysis
of multilineage hematopoietic reconstitution
in a single staining by evaluating the presence
of erythroid (CD235a+), myeloid (CD33+), B-
lymphoid (CD19+), and progenitor (CD34+/++)
cells. Such staining could be accomplished
with a modest sample of BM reserving enough
BM for other flow cytometric, molecular, or
functional analyses.
Hematopoietic stem cells are defined by
their capacity for long-term multilineage
growth potential and a capacity for self-renew-
al. Although no criteria exist to specifically
identify stem cells based on cell-surface anti-
gen expression, a stem cell phenotype has
been discerned that defines a population of
cells greatly enriched for stem cells. We detect-
ed CD34++CD133+CD38lowcells in the BM of
engrafted mice, strongly suggesting the pres-
ence of hematopoietic stem cells. The use of
multi-parameter flow cytometry to detect these
cells offers stronger evidence for stem cell
engraftment than is provided by the detection
of CD34+/++cells alone and provides the possi-
bility to enumerate, isolate, and study these
cells. Interestingly, we also observed a popula-
tion of CD34++CD133+CD38lowcells in the
Article
Figure 6. Detection of human erythrocytes in mouse hematopoietic tissues. Erythrocytes
were detected using CD235a-APC staining of BM and spleen cells. These data were gated
for PI–CD59+ events excluding mouse cells and doublets as shown in Figure 4. The per-
centages of total human CD59+events expressing low levels of forward- and side-light scat-
ter (ovals) and CD235a (rectangular regions) are indicated.
Figure 7. Detection of human myeloid cells in mouse bone marrow. (A) Expression of CD66b, CD14, and CD15 on CD33+cells. (B) Co-
expression patterns of CD66b, CD14, and CD15. Data in (A) and (B) were obtained using mouse BM harvested 229 days after transplant -
ation with human FBM and were gated for PI-CD59+events excluding mouse cells and doublets as shown in Figure 4. (C) Co-expression
patterns of CD66b, CD14, and CD15 staining of PI–human FBM. The intensity of CD66b-FITC staining is indicated by shades of green
color in all dot-plots.
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[page 8][Stem Cell Studies 2010; 1:e1]
murine liver, but not the spleen. The adult
spleen is an alternative site of hematopoiesis
in mice when BM capacity is insufficient.25
Nonetheless, the spleen does not seem to sup-
port a significant population of human stem
cells in an NSG mouse. Hematopoietic progen-
itors are known to be present in the adult
liver.26,27Our findings show that the murine
liver is a site of active extra-medullary hemato -
poiesis, as evinced by the spectrum of
CD34+/++cells observed, and suggests that the
liver may be a more important hematopoietic
organ than the spleen in an otherwise healthy
immunodeficient mouse. These findings
encourage further study of the contribution of
these liver precursors to normal and stressed
hematopoiesis.
We evaluated myeloid differentiation using
CD14 and CD15 or CD66b as markers of mono-
cytes and neutrophils, respectively, rather than
using the pan-myeloid antigen CD33.
Detection of short-lived neutrophils would be a
better indication of active hematopoiesis than
measuring a heterogenous mixture of myeloid
cells that includes longer-lived monocytes and
macrophages. CD15 and CD66b were co-
expressed in the BM of chimeric mice.
Although populations of CD14+(monocytes)
and CD66b+(neutrophils) cells were observed
in mice and FBM, additional populations of
cells were identified by these markers in
engrafted mice. CD14 expression is not exclu-
sive to the monocyte lineage and has been
reported to be weakly expressed on granulo-
cytes such as neutrophils and basophils.28
CD66b expression on neutrophils can be
increased by activation,20 and this carcinoem-
bryonic antigen is also expressed on other
granulocytes such as eosinophils29,30 and
basophils.31Thus, it appears that the mouse
BM environment causes the activation and/or
supports the development of granulocytes in
an atypical manner.
Evidence of erythropoiesis and megakary-
ocytopoiesis was present in the BM and
spleens of engrafted mice. The use of immuno -
deficient mice to study the development and
survival of these cells is of interest to investi-
gators studying gene therapy,32ex vivo cell
expansion,33,34and hematopoietic transplant -
ation.22Unfortunately, the survival of human
erythrocytes is poor in the murine circula-
tion.32We did observe CD235a+cells in the
spleen, but higher levels were detected in the
BM with evidence of active erythropoiesis.
Conversely, light-density splenocytes were
more enriched for platelets/megakaryocytes
than the BM and offer the most reliable
method of detecting these cells in engrafted
mice. Thrombocytopenia is associated with
graft failure,35so the ability to study the devel-
opment of platelets in a mouse model is a wel-
come development.
Immunodeficient mice exhibiting full
Article
Figure 8. Detection of human megakaryocytes and platelets in mouse hematopoietic tis-
sues. Data were collected from BM and light-density spleen cells gated for PI–CD59+events
and excluding mouse cells and doublets as shown in Figure 4. The percentages of total
human CD59+events expressing low levels of forward- and side-light scatter (ovals) in
addition to CD41a-FITC and CD42b-PE (rectangular regions) are indicated.
Figure 9. Detection of human B-lymphoid cells in mouse bone marrow. B-cells were pri-
marily detected using CD19-APC expressing low levels of forward- and side-light scatter
(oval gate) in addition to gating for PI–CD59+events excluding mouse cells and doublets
as shown in Figure 4. The percentage of total human CD59+events expressing CD19 (rect -
angular region) is indicated. BM cells were also co-stained with CD20-FITC, CD22-PE,
and CD34-PC7. Co-expression of these markers is shown using polychromatic dot-plots
in which CD19+events are shown in dark blue and CD19+CD20+events are shown in light
blue, as indicated in the top-right plot. Note the presence of CD19+events among CD34+
events and the predominance of CD20 expression among CD34–events.
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[Stem Cell Studies 2010; 1:e1][page 9]
human hematopoietic reconstitution are of
great interest to the study of human immunol-
ogy and methods to treat and prevent disease.
We observed B-, T-, and NK-cell development in
NSG mice. B-lymphopoiesis in the BM is gen-
erally robust in engrafted mice comprising a
large portion of the overall human hematopoi-
etic output.10Multiple stages of B-cell develop-
ment were observed in the BM, but survival
and normal function of B-cells outside the BM
is known to be attenuated in chimeric mice
because of species-restricted immune regula-
tory molecules.36We observed that develop-
ment of T- and NK-cells generally requires sev-
eral months in adult mice. The spleen was con-
sistently the best organ to detect T-cells,
whereas NK-cells were only observed in the
BM. CD4+CD8+T-cells were detected in the
spleen, and even the BM, suggesting extra -
thymic T-lymphopoiesis, albeit these cells
could have developed elsewhere and migrated
to these tissues. We further observed
CD4+CD8+T-cells in the thymus of younger
mice as previously reported,10,11but others did
not report observing a prominent double posi-
tive T-cell population in the spleen. It is pos -
sible that this cell population does not arise in
young mice and only develops in older mice in
which the thymus gland has atrophied. It
should be noted that T-cell development is
improved when neonatal mice are transplant-
ed, rather than adult mice transplanted in this
study.11Although cells with an NK-cell pheno-
type were observed in the BM, these cells were
detected infrequently and their functional sta-
tus remains unexplored. A complete under-
standing of the functional status and degree to
which lymphoid reconstitution in NSG mice
reflects the natural condition will require
much further study, and multi-parameter flow
cytometry is expected to play a salient role in
this endeavor.
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