Group Title: Journal of Translational Medicine 2006, 4:9
Title: The thrombopoietin receptor, c-Mpl, is a selective surface marker for human hematopoietic stem cells
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Title: The thrombopoietin receptor, c-Mpl, is a selective surface marker for human hematopoietic stem cells
Series Title: Journal of Translational Medicine 2006, 4:9
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Creator: Ninos JM
Jefferies LC
Cogle CR
Kerr WG
Publication Date: 38764
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Volume ID: VID00001
Source Institution: University of Florida
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Journal of Translational Medicine fioved



Research

The thrombopoietin receptor, c-Mpl, is a selective surface marker
for human hematopoietic stem cells
John M Ninos', Leigh C Jefferies2, Christopher R Cogle3 and
William G Kerr*4


Address: 1H. Lee Moffitt Cancer Center and Research Institute, Immunology Program, Department of Interdisciplinary Oncology, University of
South Florida, SRB-2, 12902 Magnolia Drive, Tampa, FL 33612-9416, USA, 2AstraZeneca LP, Drug Safety US, FOC NW2-263, Wilmington,
Delaware 19850-5437, USA, 3University of Florida, Division of Hematology/Oncology, 1600 SW Archer Road, ARB R4-252, P.O. Box 100277,
Gainesville, FL 32610-0277, USA and 4H. Lee Moffitt Cancer Center and Research Institute, Immunology Program, Departments of
Interdisciplinary Oncology and Biochemistry, University of South Florida, SRB-2, 12902 Magnolia Drive, Tampa, FL 33612-9416, USA
Email: John M Ninos ninosj@moffitt.usf.edu; Leigh C Jefferies leigh.jefferies@astrazeneca.com; Christopher R Cogle c@ufl.edu;
William G Kerr* kerrw@moffitt.usf.edu
* Corresponding author



Published: 16 February 2006 Received: 20 October 2005
journal of Translational Medicine 2006, 4:9 doi: 10.I 186/1479-5876-4-9 Accepted: 16 February 2006
This article is available from: http://www.translational-medicine.com/content/4/1/9
2006 Ninos et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Thrombopoietin (TPO), the primary cytokine regulating megakaryocyte proliferation and differentiation,
exerts significant influence on other hematopoietic lineages as well, including erythroid, granulocytic and lymphoid
lineages. We previously demonstrated that the receptor for TPO, c-mpl, is expressed by a subset of human adult bone
marrow hematopoietic stem/progenitor cells (HSC/PC) that are enriched for long-term multilineage repopulating ability
in the SCID-hu Bone in vivo model of human hematopoiesis.
Methods: Here, we employ flow cytometry and an anti-c-mpl monoclonal antibody to comprehensively define the
surface expression pattern of c-mpl in four differentiation stages of human CD34' HSC/PC (I: CD34'38--, I CD34+38dim,
II:: CD34+38+, IV: CD34dlm38+) for the major sources of human HSC: fetal liver (FL), umbilical cord blood (UCB), adult
bone marrow (ABM), and cytokine-mobilized peripheral blood stem cells (mPBSC). We use a surrogate in vivo model of
human thymopoiesis, SCID-hu Thy/Liv, to compare the capacity of c-mpl+ vs. c-mpl-- CD34+38--/dim HSC/PC for
thymocyte reconstitution.
Results: For all tissue sources, the percentage of c-mpl+ cells was significantly highest in stage I HSC/PC (FL 72 10%,
UCB 67 19%, ABM 82 16%, mPBSC 71 15%), and decreased significantly through stages II, III, and IV ((FL 3 3%,
UCB 8 13%, ABM 0.6 0.6%, mPBSC 0.2 0.1%) [ANOVA: P < 0.0001]. The relative median fluorescence intensity
of c-mpl expression was similarly highest in stage I, decreasing through stage IV [ANOVA: P < 0.0001]. No significant
differences between tissue sources were observed for either % c-mpl+ cells [P = 0.89] or intensity of c-mpl expression
[P = 0.21]. Primary Thy/Liv grafts injected with CD34+38--/dimc-mpl+ cells showed slightly higher levels of donor HLA+
thymocyte reconstitution vs. CD34+38--dimc-mpl---injected grafts and non-injected controls (c-mpl+ vs. c-mpl--: CD2+ 6.8
4.5% vs. 2.8 3.3%, CD4+8-- 54 35% vs. 31 29%, CD4--8+ 29 19% vs. 18 14%).
Conclusion: These findings support the hypothesis that the TPO receptor, c-mpl, participates in the regulation of
primitive human HSC from mid-fetal through adult life. This study extends our previous work documenting human B-
lineage, myeloid and CD34+ cell repopulation by c-mpl+ progenitors to show that c-mpl+ HSC/PC are also capable of
significant T-lineage reconstitution in vivo. These results suggest that c-mpl merits consideration as a selective surface
marker for the identification and isolation of human HSC in both basic research and clinical settings.



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Background
Elucidating the nature and biology of the primitive
pluripotent hematopoietic stem cell (HSC) has been a
major goal of research efforts in hematopoiesis. Practical
and accurate identification of primitive human HSC is
useful for both research investigation of stem cell physiol-
ogy, as well as for clinical applications, such as stem cell
transplantation, stem cell expansion and gene therapy.
Isolation of candidate human HSC allow investigations
into stem cell renewal, expansion, and key events in line-
age commitment during maturation and development.
Positive selection strategies allow transplantation of HSC
capable of long-term multilineage hematopoietic recon-
stitution, while avoiding transfer of cells responsible for
graft versus host disease in the allogeneic setting, or con-
taminating tumor cells in the autologous setting.

It is now common practice to utilize monoclonal antibod-
ies raised against an array of relevant cell surface antigens
for the identification and purification of subpopulations
of human hematopoietic stem/progenitor cells (HSC/PC)
[ 1. Historically, the surface marker most commonly used
for human HSC/PC isolation has been the CD34 antigen
[2,3]. Human hematopoietic cells expressing the CD34
antigen are enriched for HSC/PC, but represent a hetero-
geneous population, including early progenitors with
high levels of CD34 (CD34Bright) [4], and lineage-commit-
ted progenitors, with decreasing levels of CD34 as they
differentiate (CD34dim) [1,5,6]. The long-term multipo-
tent repopulating capability of CD34+ HSC/PC, which
encompasses B- and T-lymphoid, myelomonocytic, eryth-
roid, and megakaryocytic reconstitution in vivo, resides
within the CD34Bright fraction [4]. In order to more finely
delineate those CD34Bright cells which possess the recon-
stitutive and self-renewal properties of primitive HSC,
other cell surface antigens have been investigated, such as
CD38, CD50, HLA-DR, CD71, CD90, CD117, and more
recently CD133 [1] and CDCP1 [7,8].

The CD38 antigen has been useful to further identify
CD34+ HSC/PC that have begun the initial stages of line-
age commitment. CD38 is a 45 kD glycoprotein whose
function is unknown. Terstappen et al. [6] found that
expression of CD38 is an early event in the differentiation
of human CD34+ adult bone marrow (ABM) cells into the
erythroid, myeloid and lymphoid lineages, and therefore,
a useful marker for indicating early lineage commitment.
Based on the differential expression of the CD34 and
CD38 differentiation antigens, the lineage-associated
antigens CD71, CD33, CD10, CD5 and CD7, together
with forward and orthogonal light-scattering properties,
the authors developed a model for human ABM HSC/PC
differentiation to include four progressive developmental
stages I through IV. Stages I (CD34++CD38--) and II
(CD34++/+CD38+/++) included the uncommitted,


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multipotent progenitor cells; stages III (CD34+CD38+++)
and IV (CD34dimCD38+++) included the lineage-commit-
ted progenitor cells. Subsequent studies have confirmed
the basic tenets of this model, and shown that the
CD34+CD38- stage I HSC/PC are highly enriched for
pluripotent stem cell activity [9], including long-term cul-
ture-initiating cells (LTC-IC) and long-term repopulating
stem cells [5,6,10-12].

Many studies of HSC have identified factors that influence
maintenance or self-renewal of the stem cell and factors
that lead to differentiation and lineage commitment.
Thrombopoietin (TPO) is a cytokine isolated by several
groups and determined to be the primary regulator of lin-
eage-committed megakaryocyte and platelet development
[13]. However, evidence has accumulated to indicate that
TPO also exerts significant influence on other hematopoi-
etic lineages as well [14-16]. The question remained
whether TPO was affecting a broad array of lineage-com-
mitted progenitor cells, or alternatively, acting on earlier
multipotent progenitors and primitive pluripotent stem
cells.

TPO interacts with the surface receptor, c-mpl, a member
of the hematopoietic growth factor receptor superfamily
[13]. Vigon et al. [17] originally cloned and characterized
c-mpl as the human homolog of the v-mpl oncogene from
the cDNA library of the human erythroid leukemia cell
line. They noted its expression by reverse transcriptase-
polymerase chain reaction (RT-PCR) in human placenta,
bone marrow (BM), fetal liver (FL), fetal peripheral blood,
umbilical cord blood (UCB) and adult peripheral blood
(PB), and by RNA blot in murine immature hematopoi-
etic precursor cells [18]. Methia et al. [19] found that c-mpl
mRNA transcripts were expressed in purified CD34+
hematopoietic cells by RT-PCR. We subsequently found
that c-mpl is expressed at the cell surface by a subset of
murine and human HSC/PC that are enriched for cells
capable of long-term multilineage hematopoietic repopu-
lation in primary recipients [20].

In the current study, we further define the expression of
the c-mpl surface receptor in human CD34+ HSC/PC iso-
lated from four major sources of HSC from midfetal
through adult life: FL, UCB, ABM, and cytokine-mobilized
peripheral blood stem cells (mPBSC). This analysis
reveals that c-mpl receptor expression is highest on the
most primitive Stage I subset of CD34+ HSC/PC and pro-
gressively declines through Stages II, III and IV, regardless
whether the source is fetal, neonatal or adult. Moreover,
the intensity of c-mpl expression can serve to define a
primitive stage of human CD34+ HSC/PC from these four
tissue sources. In addition, we show that the c-mpl+ frac-
tion, in contrast to the c-mpl- fraction, shows a significant
increase in T-lineage thymocyte repopulation versus con-


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trol grafts in the SCID-hu Thy/Liv in vivo model of human
thymic development. These studies further indicate that c-
mpl is a selective human HSC surface receptor with a rel-
evant role in the regulation of the stem cell compartment,
and have practical implications for HSC positive selec-
tion, expansion, and transplantation protocols for both
basic research and clinical applications.

Methods
Human tissues
Anti-coagulated UCB samples were obtained from two
sources. UCB specimens were collected at the University
of Pennsylvania Health System Cord Blood Program
(Philadelphia, PA) and the Life South Cord Blood Bank of
the University of Florida (Gainesville, FL) with appropri-
ate informed consents granted by the patients. Fresh ABM
mononuclear cell specimens were obtained from Poietic
Technologies (Gaithersburg, MD) from normal healthy
volunteers with full informed consent. mPBSC were
obtained from four cancer patients who were mobilized
with a standard regimen of G-CSF (Neupogen) beginning
5 days prior to collection and twice daily during collec-
tion. mPBSC collections were performed by the University
of Pennsylvania Transfusion Medicine Section using
either COBE Spectra (Gambro BCT, Inc., Lakewood, CO)
or Fenwal CS-3000 Plus (Baxter Healthcare Co., Deerfield,
IL) blood cell separators. The four patients were being
treated with high dose chemotherapy followed by mPBSC
transplant for the following diseases: Hodgkin's Disease,
Neuroblastoma, and Non-Hodgkin's Lymphoma (2
patients). All patients consented to the use of small aliq-
uots of these cells for research purposes according to the
University of Pennsylvania Hospital and Pathology
Department guidelines effective at the time of collection.
Human fetal tissues (liver, thymus and femurs) were
obtained from Advanced Bioscience Resources (Alameda,
CA) with all appropriate and necessary informed consent
measures fulfilled.

Flow cytometric analysis of human HSC
Mononuclear cells (MNC) from FL, UCB, ABM and
mPBSC were isolated, counted on a hemacytometer, pel-
leted at 200 g for 10 minutes at 4C and resuspended in
300 ul of chilled, degassed MACS" Buffer (MB) [phos-
phate buffered saline pH 7.2, 0.5% bovine serum albu-
min, 2 mM EDTA] per 108 cells. Prior to antibody
staining, the MNC were FcR-blocked with human IgG,
then magnetically labeled with anti-CD34 coupled indi-
rectly to MACS magnetic microbeads using the Miltenyi
CD34 Progenitor Cell isolation kit (Miltenyi Biotech,
Auburn, CA) according to the manufacturer's instructions.
Following magnetic labeling, the labeled MNC cells were
washed, resuspended in 500 uL chilled MB per 108 cells
and loaded onto a Miltenyi MACS"VS+ positive selection
column and placed in the magnetic field of a Vario MACS"


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separator. The column was washed 3x with chilled MB.
The CD34+ cells remaining in the column were eluted
with 5 ml of chilled MB. The CD34+ selected cells were
counted on a hemacytometer and stained with the follow-
ing two antibody panels: 1) isotype control panel: CD34-
FITC (clone 581; 10 uL; BD Biosciences, San Diego, CA),
CD38-PE (clone HIT2; 3 uL; Caltag Laboratories, Burlin-
game, CA) and mouse IgG1-biotin (2.4 ug/24 uL; Caltag)
and 2) c-mpl panel: CD34-FITC, CD38-PE, c-mpl-biotin
(clone 3G4/CD110; 2.4 ug/5 uL; Genentech, Inc., San
Franciso, CA) [21,22]. Biotinylated antibodies were
revealed with Streptavidin-APC (3 uL/106 cells; BD Bio-
sciences). The final concentration of IgG1-biotin and c-
mpl-biotin for each experiment were equivalent. The cells
were stained at a concentration of 106 cells/50 uL staining
medium (SM) [PBS, 1% fetal bovine serum] for 30 min-
utes at 4C. The cells were washed in SM, pelleted at 300
g and resuspended in SM with either propidium iodide
(PI) [1 ug/mL; Sigma, St. Louis, MO] or 7-AAD (10 uL/
mL; BD Biosciences) for dead cell exclusion. The stained,
CD34-selected cells were then acquired on either a FACS-
Vantage" or a FACSCaliburTM flow cytometer using Cel-
1QuestTM software (BD Biosciences). Data was analyzed
using FlowJo version 4 software (Treestar Inc., Ashland,
Oregon). From both staining panels, 100,000 events were
acquired, unless sample size was limiting (UCB: 7,000-
38,000 events; FL 40,000-100,000 events). The analysis
protocol was consistent and uniform across all samples
from each HSC source. Minor adjustments to the gates
were made between individual specimens to account for
experimental variability. Beginning with the cells stained
with the isotype control panel, a "cell" gate was created on
a forward scatter (FSC) vs. side scatter (SSC) plot to elim-
inate debris, platelets, red cells, cell doublets, and MNC
with high SSC. From within the "cell" gate, a "viability"
gate was applied on a log histogram plot of PI or 7-AAD
to exclude nonviable events. A CD34-FITC vs. CD38-PE
contour plot was then selected on viable cells. From this
plot, four HSC/PC differentiation stage subsets based on
CD34 vs. CD38 expression were delineated as per Terstap-
pen et al. [6]: (I): CD34+CD38- (II): CD34+CD38dim (III):
CD34+CD38+ (IV): CD34dimCD38+. From within each
"CD34/CD38 stage" of the isotype control-stained cells, an
IgG1-APC histogram plot was created. The "c-mpl-" gate
was then defined by including 99.5% of the IgG1-APC
negative events. This gate was adjusted by + 0.1-0.2%
when the FlowJo software could not select exactly 99.5%
of the events. Conversely, a "c-mrpl+" gate was defined to
include 0.5% ( 0.1-0.2%) of the IgG1-APC positive
events and extended to the far right of the APC histogram.
The "cell", "viability", "CD34/CD38 stage" and "c-mpl+"
gates were then applied exactly, without alteration, to the
paired cell sample stained with the c-mpl antibody panel
to determine the percentage ofc-mpl+ cells in each CD34/
CD38 stage. Two statistical methods in the FlowJo soft-


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ware super-enhanced Dmax subtraction (SED) [23] and
Overton subtraction [24] were also used to determine
the percentage of c-mpl+ events relative to the isotype con-
trol in a univariate population comparison. Furthermore,
the median fluorescence intensity (MFI) of the log APC
parameter for each CD34/CD38 stage from each specimen
was determined for both the IgG1 isotype control and c-
mpl panel-stained sample pairs. The MFI of the c-mpl
panel-stained cells was then normalized against the MFI
of the IgG1 isotype control panel-stained cells for each
CD34/CD38 subset to allow comparison of the Relative
MFI (RMFI) across each specimen and each stem cell tis-
sue source. Next, for each sample from every tissue source,
we again selected those events falling within both the
"cell" gate and the "viable" gate to include only viable
MNC. We analyzed these viable MNC for c-mpl-APC flu-
orescence intensity on a histogram plot of the log APC
parameter. We then selected three discrete subsets of cells
based solely on their percentile level of c-mpl-APC fluo-
rescence intensity for further analysis: (1) High [98-
100%tile] (2) Intermediate [48-50%tile] and (3) Low [18-
20%tile]. These three percentile groups were then analyzed
on CD34-FITC vs. CD38-PE contour and dot plots to dis-
cern qualitative differences in CD34 and CD38 expres-
sion. A similar analysis was then performed on a select
number of specimens for a more complete range of High
percentile groupings by drawing five gates on the histo-
gram plot to define a progression of five discrete levels of
High c-mpl expression: 79-80%tile, 84-85%tile, 89-
90%tile, 94-95%tile, and 99-100%tile.

Statistical comparisons were performed using Prism 4.0
software (GraphPad Software, San Diego, CA). By conven-
tion, the threshold value, a, was set to 0.05. The percent-
age of viable c-mpl+ cells was determined by: 1) manual
gating, 2) the SED algorithm, and 3) the Overton subtrac-
tion algorithm. For each human tissue studied (FL, UCB,
ABM, mPBSC), the following human HSC/PC subset
groups were compared in a repeated measures two-way
analysis ofvariance: (I) CD34+CD38-, (II) CD34+CD38dim,
(III) CD34+CD38+, and (IV) CD34ditCD38+. Tukey's and
Bonferroni's Multiple Comparison post-tests were then
performed to compare pairs of group means. In addition,
a post-test for a linear trend was performed with the sub-
set groups arranged in the following order: I, II, III, IV.
Next, the RMFI values (after normalizing to the isotype
control MFI) were analyzed in the same manner and using
the same groups for comparison as for the c-mpl+ analyses
described above.

Generation of SCID-hu ThylLiv mice
Eight week old C.B-17 scid/scid severe combined immune
deficiency (SCID) mice were implanted with human fetal
thymus and liver fragments as previously described [25-
28]. Briefly, human fetal thymus and liver tissue were


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obtained from 18-22 week gestational fetuses. During the
preparation of the tissue grafts, MNC were collected and
analyzed by flow cytometry using a panel of human leu-
kocyte antigen (HLA) antibodies (One Lambda, Inc.,
Canoga Park, CA) and found to be negative for HLA-B8.
The 8 week old SCID mice were anesthetized with Keta-
mine. The fur over the mouse kidney was shaved and the
skin prepped with alcohol and betadine. The skin and per-
itoneum were opened over the right kidney. The kidney
was exposed with a hemostat. Two ~1 mm3 cubes of fetal
liver were placed adjacent to one ~ 1 mm3 cube of fetal thy-
mus in a 16 gauge trocar and co-implanted as a unit under
the renal capsule. The peritoneal and fascial layers were
restored with sutures (Ethicon, Somerville, NJ), and the
skin incision was secured with MikRon 9 mm Autoclips
(MikRon Precision, Gardena, CA). The human Thy/Liv
transplants were allowed to engraft for 8 weeks.

Analysis of engraftment in SCID-hu ThylLiv grafts
Bone marrow MNC from an HLA-B8+ normal volunteer
donor were positively selected for the CD34 antigen with
CD34 immunolabeled magnetic beads using the CD34
Progenitor Cell kit (Miltenyi Biotech). The CD34+ cells
were stained with CD34-FITC, CD38-PE, and biotinylated
anti-c-mpl (clone 3G4/CD110) [21,22]. A small aliquot
of the sample was stained with CD34-FITC, CD38-PE and
biotinylated IgG1-isotype control. The biotinylated anti-
bodies were revealed with Streptavidin-APC. The cells
were washed and resuspended in SM containing PI (1 ug/
mL). Samples were acquired and sorted on a FACSVan-
tage'T flow cytometer using CellQuestTM software. After gat-
ing for viable cells based on PI exclusion and light side
scatter, an IgG1-APC histogram for CD34+CD38--/dim cells
was plotted. A c-mpl-- gate was drawn to include 99% of
the cells in the IgG1 isotype control stain. A c-mpl+ gate
was drawn from the end of the c-mpl-- gate to the far right
of the histogram. These two gates were then applied to the
c-mpl-panel stained cells, and CD34+CD38--/dimc-mpl+
and CD34+CD38--/dimc-mpl-- cells were sorted into tubes
containing RPMI1640 (Mediatech, Inc., Herndon, VA)
with 3% fetal bovine serum and 10 mM HEPES. The
sorted cells were counted on a hemacytometer, pelleted at
200 g for 10 minutes at 40C and resuspended in sterile
PBS. The diluted CD34+CD38--/dimc-mpl+ and
CD34+CD38--/dimc-mpl-- cells were loaded separately into
a Hamilton syringe. SCID-hu Thy/Liv mice were irradiated
with 400 rads from a Cs137 source prior to injection. The
mice were anesthetized with ketamine, and the Thy/Liv
grafts were gently exteriorized and inspected. Only mice
with healthy, robust grafts (numbering 22) were chosen
for HSC subset injections. Grafts were injected with the
viable sorted HSC subsets at 30,000 or 60,000 cells per
graft. Ten grafts were injected with 60 k (3 grafts) or 30 k
(7 grafts) of viable CD34+CD38--c-mpl-- cells. Nine grafts
were injected with 60 k (2 grafts) or 30 k (7 grafts) of via-


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A FL UCB ABM mPBSC
Iv vIv
a. Iv III III Ill
A I II IV
0 IV
0 I *i II -

i . 1 1 o'0 21o I 1 o 1' 2 1 3n 1 1 l I 2 I 3 0 .n I Il 1 21 I 3 In4
4- CD34-FITC

B 4 I IgG1Ctl.
E IV
Smill




4 c-MPL-APC -
S18-20%tile c-mpl-APC
48-50%tile c-mpl-APC
Te ., p. -O 98-100%tile c-mpl-APC




ct CD34-FITC . p f

D 18-20%tile c-mpl-APC
I IO 48-50%tile c-mpl-APC
a 98-100%tile c-mpl-APC
0



41- CD34-FITC


The most primitive CD34+ HSCIPC from all major tissue sources express the highest levels of c-mpl surface
receptor. A. Representative CD34-FITC vs. CD38-PE contour plots generated from magnetically selected viable CD34'
mononuclear cells from representative specimens of the four major tissue sources of human HSC/PC: FL, UCB, ABM, and
mPBSC. Gates defining the four differentiation stages of human CD34+HSC/PC as defined by Terstappen et al. [6] are shown:
I: CD34+38-. II: CD34+38dm IIll: CD34+38+ IV: CD34dim38+. B. Overlay of the c-mpl-APC histogram plots of viable human
CD34+ mononuclear cells from the four HSC/PC differentiation stages depicted in Fig. IA above. Stage I [red]. Stage II [blue].
Stage III [green]. Stage IV [light blue]. For comparison, a control histogram plot [gray] is shown that is generated from differen-
tiation stage I cells stained with an IgG I isotype control-APC antibody equivalent in concentration to the c-mpl-APC antibody
stain. Isotype control histogram plots for each HSC/PC differentiation stage were used to separately set the manual gates that
define the c-mpl+ and c-mpl-- cell populations for each stage. C. CD34-FITC vs. CD38-PE dot plots derived from the same rep-
resentative human CD34+ tissue specimens as depicted in Fig. IA. From each specimen, all cell events were initially categorized
by their percentile level of fluorescence on the c-mpl-APC parameter on a log histogram plot. Three gates were drawn on the
histogram plot to define three discrete levels of c-mpl expression: High (98-100 percentile); Intermediate (48-50 percentile);
Low (18-20 percentile). Viable cell events falling within one of these three percentile ranges are depicted on the dot plot with
their corresponding color code: High [light blue]; Intermediate [dark blue]; Low [red]. D. Same data as C, but using contour
plots to visualize the data. High [red]; Intermediate [yellow]; Low [blue].







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Journal of Translational Medicine 2006, 4:9


ble CD34+CD38--c-mpl+ cells. Three grafts were reserved
as non-injected controls. The c-mpl+ and c-mpl---injected
ABM stem cell subsets were then allowed to repopulate
the Thy/Liv grafts for 8 weeks. After 8 weeks, surviving
mice were: c-mpl+: 60 k (1), 30 k (3); c-mpl--: 60 k (2), 30
k (5); non-injected: (3). The Thy/Liv grafts from the sur-
viving mice were carefully exteriorized and removed. Sin-
gle cell suspensions from each graft were prepared and
stained separately with: (1) IgG1-FITC (5 uL/106 cells; BD
Pharmingen), IgG1-PE (5 uL/106 cells; BD Pharmingen),
IgG2b-biotin (10 uL/106 cells; Caltag), (2) CD34-FITC
(10 uL/106cells; BD Pharmingen; clone 581), CD2-PE (10
uL/106 cells; BD Pharmingen; clone RPA-2.10) and HLA-
B8-biotin (5 uL/106 cells; One Lambda), and (3) CD4-
FITC (10 uL/106 cells; BD Pharmingen; clone RPA-T4),
CD8-PE (10 uL/106 cells; BD Pharmingen; clone RPA-T8),
and HLA-B8-biotin (5 ul/106 cells). Biotin conjugates
were revealed with Streptavidin-APC (3 uL/106 cells; BD
Pharmingen). Cells were resuspended in SM with PI (1
ug/mL) for dead cell exclusion. For each antibody panel,
100,000 events were acquired on a FACSCalibur'M flow
cytometer using CellQuestTM software. Analysis was per-
formed using FlowJo version 4 software. A "human cell"
gate was drawn around the human lymphoid cell popula-
tion based on a FSC vs. SSC plot. A "viable" gate was cre-
ated around viable cells on a PI vs. FSC plot. The viable
human lymphoid cell population in the non-injected
Thy/Liv grafts was used to define the HLA-B8 positive and
negative gates for both the CD34/CD2/HLA-B8 and CD4/
CD8/HLA-B8 antibody stain panels. In each case, an HLA-
B8-- gate was defined to include 99.0% of the HLA-B8-
stained non-injected Thy/Liv grafts, and an HLA-B8+ gate
was drawn to extend from the HLA-B8- gate to the
extreme right of the APC parameter log histogram plot.
Subsequent analysis (data not shown) confirmed that this
strategy for defining the HLA-B8+ events was more restric-
tive than the strategy of defining HLA-B8+ events using an
IgG2b-APC isotype control.

For statistical comparisons of Thy/Liv reconstitution, the
threshold value, a, was set to 0.05. The following thymo-
cyte subsets were analyzed: total CD2+, CD4+CD8+,
CD4+CD8--, and CD8+CD4--. For each subset, the follow-
ing three SCID-hu Thy/Liv graft injection groups were
compared for the percentage of viable HLA-B8+ lymphoid
cells in a one-way analysis of variance: (1) non-injected con-
trol Thy/Liv grafts; (2) CD34+CD38--idic-mpl---injected
Thy/Liv grafts and (3) CD34+CD. "..--mpl+-injected
Thy/Liv grafts. A Dunnet's Multiple Comparison post-test
was performed to compare separately the c-mpl- and c-
mpl+ injections with the non-injected control Thy/Liv
grafts. In addition, a post-test for a linear trend was per-
formed with the groups arranged in the following order:
non-injected controls, CD34+CD38--/dimc-mpl---injections, and
CD34+CD38--/dimc-mpl+-injections.


http://www.translational-medicine.com/content/4/1/9



Results
C-mpl is expressed by primitive HSC from all major tissue
sources
Our previous findings demonstrated that c-mpl is
expressed by the subset of CD34+ cells in normal human
ABM that lack or have low surface expression of CD38
[20]. The CD34+CD38--/dim population of cells is known
to be enriched for primitive HSC relative to the
CD34+CD38+ subset based on its ability to support long-
term multilineage repopulation in vivo in a pre-immune
fetal sheep model [5] or to give rise to significant numbers
of LTC-IC in vitro [11,12]. Therefore, we conducted a more
comprehensive and extensive examination of c-mpl recep-
tor surface expression in distinct human hematopoietic
CD34/CD38 HSC/PC subsets. In order to characterize
more completely the expression pattern of c-mpl in
human CD34+ cells, we chose to investigate c-mpl expres-
sion across four different human HSC/PC differentiation
stages originally characterized by Terstappen et al. [6]: (I)
CD34+CD38-. (II) CD34+CD38dim (III) CD34+CD38+
(IV) CD34dimCD38+. We analyzed the expression of the c-
mpl receptor in these four stages of HSC/PC differentia-
tion from four major sources of human HSC: FL, UCB,
ABM and mPBSC. These four tissue sources represent the
major locations of human hematopoiesis from midfetal
life through adulthood. In each case, we isolated CD34+
cells by magnetic bead positive selection, and stained
equivalent numbers of these cells with two monoclonal
antibody panels: (1) Isotype control panel: monoclonal
antibodies to CD34-FITC, CD38-PE and IgG1-APC or (2)
c-mpl panel: monoclonal antibodies to CD34-FITC,
CD38-PE and c-mpl-APC. Gates were set to identify the
four stages of HSC/PC differentiation as defined by Ter-
stappen et al. [6] (Figure 1A). In each case, the analysis
was first defined by the isotype control antibody panel-
stained cells, and then applied without modification to
the c-mpl antibody panel-stained cells. Representative c-
mpl histograms for each CD34/38 population from each
tissue are shown in Figure lB.

Using this manual gating strategy, the percentage of c-
mpl+ cells in stage I was >50% for every sample tested
except one (UCB#2 = 44.5%), and ranged from 44.5% to
94.5% (ABM#1) [Table 1]. For every sample tested, the
percentage of c-mpl+ cells decreased uniformly as the dif-
ferentiation stages progressed from I through IV (Figure
2A). The mean percentage ( standard deviation [S.D.]) of
c-mpl+ cells in stage I ranged from 67.18% + 18.55%
(UCB) to 81.96% + 16.29% (ABM), while the mean per-
centage ( S.D.) of c-mpl+ cells in stage IV ranged from
0.17% + 0.07% (PBSC) to 7.91% 13.43% (UCB). A
repeated measures two-way analysis of variance compar-
ing the two variables, (1) tissue source and (2) differenti-
ation stage, indicated that the differences observed in the
percentage of c-mpl+ cells for the four differentiation


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Table I: Percentage of c-mpl* cells in human HSC/PC differentiation stages

PERCENTAGE c-mpl+


Stage I


Stage II


Stage Ill Stage IV


Tissue Source #


FL 5 Mean
S.D.
UCB 5 Mean
S.D.
ABM 5 Mean
S.D.
mPBSC 4 Mean
S.D.


Manual Overton SED Manual Overton SED Manual Manual
Gate Algorithm Algorithm Gate Algorithm Algorithm Gate Gate


71.54
10.48
67.18
18.55
81.96
16.29
71.40
14.55


77.23
8.02
76.24
11.11
85.66
13.06
77.58
13.25


86.43
5.94
83.82
9.55
92.25
9.69
85.05
9.29


50.76
14.94
49.92
21.59
61.60
23.94
48.25
22.86


60.19
12.04
65.83
19.74
69.87
21.76
57.14
19.74


72.09
11.24
75.63
18.91
80.06
19.37
66.85
17.39


10.15
5.99
12.85
12.51
5.93
3.93
7.87
4.53


3.40
3.49
7.91
13.43
0.58
0.61
0.17
0.07


ABM, adult bone marrow; FL, fetal liver; mPBSC, cytokine-mobilized peripheral blood stem cells; SD, standard deviation; SED, super-enhanced
Dmax subtraction; UCB, umbilical cord blood


stages were highly significant (p < 0.0001) [Table 2]. Fur-
thermore, a post-test for a linear trend from stage I
through stage IV was significant for each tissue source (P
< 0.0001), indicating a significant decreasing linear trend
for the percentage of c-mpl+ cells successively in the order
of stage I, II, III and IV. This analysis also showed that the
greatest decrease in the mean percentage of c-mpl+ cells
between contiguous stages occurred between stages II and
III for each tissue source with a mean difference ranging
from 37.1% (UCB) to 55.7% (ABM). Bonferroni post-
tests individually comparing pairs of contiguous stage
means (I vs. II, II vs. III, III vs. IV) showed that the differ-
ences were significant for all tissue sources for stages I vs.
II (p < 0.05: UCB & ABM; p < 0.01: FL & mPBSC) and
stages IIvs. III (p < 0.001), but not significant for stages III
vs. IV. Thus, the greatest and most significant loss of c-mpl
expression during this differentiation scheme is associated


with the acquisition of high level CD38 expression by
HSC/PC. However, there was no significant interaction
between the tissue source and the differentiation stage val-
ues (P = 0.8918). Furthermore, the overall effect of the tis-
sue source on % c-mpl+ was not significant (P = 0.2949)
[Figure 2B].

We confirmed these findings using two mathematical
comparison algorithms that provide the percentage of
events that are positive compared to the control popula-
tion. The Overton cumulative histogram substraction
algorithm [24] was approximately 4 to 9 percentage
points higher than the manual gating method for stage I,
ranging from 76.24% + 11.11% (UCB) to 85.66% +
13.06% (ABM). The stage I mean % c-mpl+ calculated by
the super-enhanced Dmax subtraction (SED) algorithm
[23] was approximately 10-17 percentage points higher


Table 2: Repeated Measures Two-way ANOVA of c-mpl expression in Human HSC/PC: Stage vs. Tissue Source


Stage Tissue Interaction


FL
UCB
ABM
mPBSC


Bonferroni's Multiple Comparison


0.2949 Stage I vs. II Stage II vs. Ill Stage III vs. IV


FL
UCB
ABM
mPBSC


FL
UCB
ABM
mPBSC


<0.01
<0.05
<0.05
<0.01


<0.001
<0.001
<0.001
<0.001 I


0.0779 Stage I vs. II Stage II vs. Ill Stage III vs. IV


FL
UCB
ABM
mPBSC


<0.05
<0.01
<0.00 I
<0.001


Linear Trend (One-way)

Order: 1-11-111-IV

<0.0001
<0.0001
<0.0001
<0.000 I

Order: 1-11-111-IV

<0.000 I
<0.00 I
<0.0001
<0.0001


Values represent calculated p values for the indicated analyses. ABM, adult bone marrow; ANOVA, analysis of variance; FL, fetal liver; HSC/PC,
hematopoietic stem cells/progenitor cells; mPBSC, cytokine-mobilized peripheral blood stem cells; NS, not significant (p > 0.05); RMFI, relative
median fluorescence intensity; UCB, umbilical cord blood


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% c-mpl+ <0.0001 0.8918


c-mpl RMFI <0.000 I 0.2102


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A


+_
a.
E
6











C


100-
90.
80-
70-
60-
60-
40-
30-
20-
10-

FL UCB ABM mPBSC
Human Tissue Source




100-


10-






0.1
FL UCB ABM mPBSC
Human Tissue Source


B


+_
a.
E
6









-
D


=II

17-1111
17- IVV
















IV


HSCIPC CD34CD38 Stage


Figure 2
The expression of c-mpl is significantly higher in stage I CD34" HSC/PC and decreases with advancing differen-
tiation stage. A&B. For each specimen from every tissue source, CD34+ cells were plotted with FlowJo software on a
CD34-FITC vs. CD38-PE contour plot, and gates demarcating HSC/PC differentiation stages I through IV [6] were applied (as
depicted in Fig. I A). Cells within each stage were stained with an IgG I isotype control-APC antibody equivalent in concentra-
tion to the c-mpl-APC antibody used. On a log histogram plot of IgG I-APC fluorescence, a c-mpl-- gate was manually drawn to
include the left-most 99.5% ( 0.2%) of isotype control-stained cells. A c-mpl' gate was manually defined to extend from the
end of the c-mpl-- gate to the far right of the histogram plot. These gates were defined separately for each HSC/PC differentia-
tion stage for each tissue source, and then applied without alteration to their corresponding c-mpl-APC stained cells within
each stage from each tissue. The percentage of cells falling within the c-mpl+ gate was then calculated by the FlowJo software.
Error bars = standard error of mean (SEM). C&D. For every specimen, the median fluorescent intensity (MFI) of viable
CD34+ cells from each differentiation stage was calculated by FlowJo software from the log histogram plot of the c-mpl-APC
fluorescence parameter. These MFI values for each stage of each tissue specimen were then normalized individually with the
appropriate MFI value of the corresponding differentiation stage HSC/PCs stained with the IgG I isotype control, yielding the
Relative MFI (RMFI). Error bars = SEM.


than the manual gating method, ranging from 83.82% +
9.55% (UCB) to 92.25% + 9.69% (ABM). Likewise, the %
c-mpl+ for each sample as determined by either the Over-
ton or SED subtraction algorithms decreased uniformly as
the differentiation stages progressed from stage I through
IV.

In order to quantify differences in the intensity of c-mpl
receptor expression between the four differentiation
stages of each sample, we calculated the MFI of c-mpl-APC


staining on a histogram plot of the APC parameter using
a log scale. For each differentiation stage of each sample,
we similarly calculated the MFI of the isotype control-APC
on the log scale histogram plot. We then divided the c-
mpl MFI by the isotype control MFI to derive a Relative
MFI (RMFI) normalized to the isotype control staining
intensity.

In every sample except two (UCB#3 & UCB#5), the RMFI
was highest in differentiation stage I and decreased uni-


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HSC/PC CD34CD38 Stage


MFL
MUCB
MABM
mPBSC












MFL
MUCB
MABM
mPBSC


Journal of Translational Medicine 2006, 4:9








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Table 3: Relative MFI ofc-mpl expression in human HSC/PC differentiation stages


Relative Median Fluorescence Intensity


Tissue Source


FL

UCB

ABM

mPBSC


Stage I


5 Mean
S.D.
5 Mean
S.D.
5 Mean
S.D.
4 Mean
S.D.


7.13
2.06
13.14
7.77
22.25
18.32
26.55
36.08


Stage II

3.68
1.15
8.38
6.04
9.04
6.12
5.72
5.01


Stage III

1.39
0.40
2.71
2.03
1.34
0.38
0.94
0.29


Stage IV

1.00
0.56
1.92
1.16
0.97
0.24
0.47
0.14


ABM, adult bone marrow; FL, fetal liver; mPBSC, cytokine-mobilized peripheral blood stem cells; SD, standard deviation; MFI, median fluorescence
intensity; HSC/PC, hematopoietic stem cells/progenitor cells; UCB, umbilical cord blood


formly through stages II, III and IV (Table 3). In UCB#3,
the RMFI was slightly higher in stage II (16.69) than stage
I (14.44). In UCB#5, the RMFI was slightly higher in stage
IV (3.48) than stage III (2.37). The mean RMFI in stage I
ranged from 7.13 2.06 (FL) to 26.55 + 36.08 (in PBSC).
The mean RMFI for each tissue source decreased uni-
formly from differentiation stage I through stages II, III
and IV (Figure 2C). Because these data were derived from
a logarithmic scale, the values were transformed using the
following equation, Y=log(Y), to convert to a guassian dis-
tribution for statistical analysis. A repeated measures two-
way analysis of variance, comparing each differentiation
stage and each tissue source, was performed (Table 2).
This analysis confirmed that the differentiation stage had
an extremely significant effect on c-mpl-APC RMFI when
considering all tissue sources studied (P < 0.0001). For
each tissue source, a post-test for a linear trend was signif-
icant for a decreasing trend from differentiation stage I
through stages II, III and IV. A Bonferroni Multiple Com-
parison post-test was calculated for each tissue source to
individually compare each stage with every other stage.
The difference in c-mpl RMFI between stage III and stage
IV was not significant for any tissue source. The difference
in RMFI between stage I and stage II was significant for the
mPBSC tissue source only (p < 0.01). All other compari-
sons between the stages were significantly different in all
four tissue sources studied [I vs. III, I vs IV, and II vs IV: all
p < 0.001; II vs III: p < 0.001 (ABM&mPBSC), p < 0.01
UCB, p < 0.05 FL]. Similar to the % c-mpl+ results, the larg-
est contiguous decrease in the mean RMFI (transformed
data) for each tissue source occurred in the transition from
stage II to stage III. Thus, the greatest log decrease in c-mpl
receptor density is correlated with strong expression of the
CD38 surface antigen. However, the interaction between
the tissue source and all values of the differentiation stage
was not significant (P = 0.0779). Furthermore, the overall
effect of the tissue source on c-mpl-APC RMFI was not sig-
nificant (P = 0.2102) [Figure 2D].


We also sought to determine whether those individual
cells expressing the very highest levels of c-mpl receptor,
as measured by fluorescence intensity of the log APC
parameter, were clustered together within a discrete
group, when considering the parameters CD34-FITC and
CD38-PE. Furthermore, we sought to determine whether
individual cells expressing the lowest levels of c-mpl
receptor could similarly be characterized in terms of
CD34-FITC and CD38-PE. This would allow us to deter-
mine, in a qualitative sense, whether the density of expres-
sion of the c-mpl receptor on an individual cell could
define that cell in terms of CD34 and CD38 antigen
expression. In every sample studied, virtually all of the
cells falling within the 98-100%tile c-mpl-APC fluores-
cence intensity were clustered among those cells express-
ing the highest levels of CD34 and the lowest levels of
CD38 (Figure 1C&D). This was true regardless of the tis-
sue source of the HSC/PCs. Not surprisingly, these high
intensity c-mpl expressing cells had a narrowly defined
low FSC and low SSC, with minimal spread (data not
shown). The cells falling within the 18-20%tile of c-mpl-
APC fluorescence intensity were more loosely clustered
among those cells expressing a wide range of levels of
CD34 surface antigen, from low to moderate to high, as
well as a range of CD38 surface antigen, from moderate to
very high, reflecting the inherent heterogenity of this pop-
ulation. The vast majority of these low intensity c-mpl-
expressing cells expressed high to very high levels of CD38
surface antigen. Interestingly, few of these low intensity c-
mpl-expressing cells expressed very low levels of CD38
surface antigen. Among the cells that expressed high
CD38 antigen density, c-mplBright cells were very rare. In
other words, there was very little overlap between the high
intensity and low intensity c-mpl stained cells on a CD34-
FITC vs. CD38-PE contour plot, and virtually no overlap
between the two populations at both the high and low
extremes of CD38 surface antigen expression. This was
true regardless of the tissue source of the HSC/PCs stud-


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Journal of Translational Medicine 2006, 4:9


ied. As expected, the FSC and SSC profile of these low
intensity c-mpl stained cells exhibited a wider and higher
range of scatter than the high intensity c-mpl stained cells
(data not shown). Not surprisingly, those cells expressing
an intermediate level of c-mpl surface receptor expressed
a broad range of CD34 and CD38 levels that fell within
the extremes displayed by the high and low intensity c-
mpl stained cells. Importantly, these intermediate level c-
mpl receptor-expressing cells did not overlap with the
high level c-mpl expressors at the lowest range of CD38
antigen expression. In fact, the analysis of a select number
of specimens for a more comprehensive series of High
intensity c-mpl expressors (79-80%tile, 84-85%tile, 89-
90%tile, 94-95%tile, and 99-100%tile) indicated that the
CD34+ cells that express the very lowest levels of CD38
antigen were nearly exclusive to the cells with the very
highest intensity c-mpl-expression (Figure 3).

The CD34+CD38-Idimc-mpl+ subset is capable of thymic
reconstitution
One limitation of the SCID-hu bone model of human
hematopoiesis [20] is that it lacks the thymic microenvi-
ronment necessary for the development and maturation
of the T-lymphoid compartment. In order to compare the
T-lymphoid reconstitution potential of human
CD34+CD38--/dimc-mpl+ cells versus that of CD34+CD38--
/dimc-mpl- cells, we used the SCID-hu Thy/Liv model of
human hematopoiesis [25-28]. Thy/Liv grafts were
directly injected with 60,000 or 30,000 HLA-disparate
human ABM cells of the CD34+CD38--/dimc-mpl+ or the
CD34+CD38--/dimc-mpl-- phenotype. Three Thy/Liv grafts
were set aside as non-injected controls. The injected cells
were allowed to repopulate the Thy/Liv grafts for 8 weeks.
After 8 weeks, the Thy/Liv grafts were harvested and single
cell suspensions prepared. Donor repopulation (HLA-B8)
of the thymic compartment was assessed by flow cytome-
try with the following antibody panels: (1) CD34-FITC,
CD2-PE, and Donor HLA-B8-APC; and (2) CD4-FITC,
CD8-PE, and Donor HLA-B8-APC (Figure 4A-F). Overall,
the CD34+CD38--/dimc-mpl+ injected cells showed donor-
derived T-lymphoid repopulation in 75% (3 out of 4
grafts) of the mice, whereas the CD34+CD38--/dimc-mpl--
injected cells, demonstrated T-lymphoid repopulation in
57% (4 out of 7 grafts) of the mice. The mean percentage
contribution of donor-derived cells to the stages of
human T-cell development were also compared for grafts
injected with CD34+CD38--/dimc-mpl+ or CD34+CD38--/
dimc-mpl-- versus the non-injected control grafts (Table 4).
For the early stage thymocytes (double positive:
CD4+CD8+), both injected populations showed very low
HLA-B8 expression levels that were not significantly dif-
ferent from the non-injected controls. Presumably, low
HLA expression in these immature thymocytes limited
our ability to discern the identity of donor cells in this
population. For the total (CD2+) and the mature (single


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positive: CD4+CD8--, CD4--CD8+) T-cell development
stages, the mean percentage contribution of HLA-B8+
donor repopulation was higher in the CD34+CD38--/dim-
mpl+ injected grafts than in the grafts that received
CD34+CD38--/dimc-mpl-- cells (Figure 5). A one-way analy-
sis of variance was performed for each T-cell subset com-
paring the CD34+CD38--/dimc-mpl+ and CD34+CD38--/
dimc-mpl---injected grafts with the non-injected grafts
(Table 5). This analysis found the means of the three treat-
ment arms to be significantly different in the CD2+,
CD4+CD8--, and CD4--CD8+ subsets (P < 0.05). A Dun-
nett's Multiple Comparison post-test was then performed
for the subsets showing significant differences, comparing
separately the c-mpl+ and c-mpl---injected grafts with the
non-injected control grafts. For these subsets, the differ-
ence in means between the c-mpl+-injected grafts and the
control grafts was significant (P < 0.05), whereas the dif-
ference in means between the c-mpl---injected grafts and
the control grafts was not significant (P > 0.05). Further-
more, a post-test for a linear trend from non-injection
control to c-mpl-- injections to c-mpl+ injections was sig-
nificant (P < 0.01). Thus, the increase in the mean per-
centage of donor-derived cells noted for each subset when
moving in the order of control grafts to c-mpl---injected
grafts to c-mpl+-injected grafts appears meaningful. This
analysis found no significant difference in the degree of
donor repopulation between injections of 60,000 or
30,000 cells.

Discussion
The marker of choice in human clinical HSC/PC selection
strategies has generally been CD34 [3,29-32]. However,
clinical selection strategies utilizing other potentially
more selective HSC markers, such as CD133 [1] or
CDCP1 [7], have been proposed. While the consensus
phenotype of primitive human HSC has been CD34Bright
CD38--/dim HLA-DR--/dim CD90+ CD117+ Lineage-- and
rhodaminel2310, this report indicates that c-mplHi/Bright
may reasonably be included as a defining characteristic of
primitive human CD34+ HSC. We found for all tissue
sources that c-mpl expression is highest in the most prim-
itive category of CD34+ HSC/PC defined by Terstappen et
al. [6], then decreases significantly as the HSC/PC differ-
entiation stage matures, manifested by increasing levels of
the CD38 differentiation antigen. The greatest and most
significant decrease in % c-mpl+ cells occurs from stage II
to stage III for all tissue sources. Similarly, the greatest
decrease in c-mpl antigen levels, as measured by Relative
MFI, occurs from stage II to stage III, indicating a close
inverse relationship between c-mpl and CD38 antigen
expression.

One potential advantage of using c-mpl as an identifier of
human HSC is the known biologic role of its ligand part-
ner, TPO. Borge et al. [33] observed that TPO promoted


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89-90%tile


ABM


LU
a



mPBSC
IF


94-95%tile


99-100%tile


Figure 3
The intensity of c-mpl expression can define the most primitive HSC. CD34-FITC vs. CD38-PE smoothed pseudo-
color dot plots derived from the same representative human CD34 ABM and mPBSC specimens as depicted in Fig. IA. From
each specimen, all cell events were initially categorized by their percentile level of fluorescence on the c-mpl-APC parameter
on a log histogram plot. Five gates were drawn on the histogram plot to define a progression of five discrete levels of High
intensity c-mpl expression: 79-80, 84-85, 89-90, 94-95, and 99-100 percentiles. Viable cell events falling within each percen-
tile range are depicted on the corresponding smoothed dot plot.


the viability of 51% of multifactor-responsive human
CD34+CD38--ABM HSC/PC, 75% of which remained via-
ble in the absence of detectable cell proliferation. This was
significantly higher than that of IL-3 (25%), c-kit ligand
(14%), or Flt-3L (14%). In contrast to the potent ability of
TPO to promote viability of CD34+CD38-- HSC/PC, TPO
did not promote the viability of CD34+CD38+ HSC/PC.
Up to 38% of the individual CD34+CD38- HSC/PC sur-
viving in 116 hours of serum-free preincubation medium
containing a single cytokine, TPO, were multipotent, gen-
erating myeloid, erythroid, and mixed myeloid/erythroid
colonies in methylcellulose containing a 10-cytokine
cocktail. Although the studies did not assay for the lym-
phoid compartment, our results suggest that these TPO-
responsive CD34+CD38- single cells possess both B-lym-
phoid [20] and T-lymphoid potential as well.

Previously, using SCID-hu Bone repopulation assays, we
showed that the capacity for CD34+ HSC/PC repopulation
correlates with the c-mpl+ fraction of CD34+CD38--
human ABM HSC [20]. These results are consistent with
the work of Petzer et al. [12] who showed that TPO could
expand LTC-IC using an in vitro assay to detect very prim-
itive HSC/PC. Borge et al. [33] subsequently demon-
strated that single CD34+CD38- cells pre-incubated in
serum-free medium containing TPO alone for 116 hours
retained nearly all [96%] of the LTC-IC capacity present in
single CD34+CD38--cells freshly placed in the human BM
stroma LTC-IC culture assay. Interestingly, while primitive


human HSC/PC have been posited to express low levels of
c-kit, significantly more LTC-IC survived in the presence
of TPO alone than in the presence of c-kit ligand alone.
The authors also observed that, in contrast to TPO, most
of the viability-promoting effect of the IL-3, c-kit ligand,
or Flt-3L cytokines individually on CD34+CD38-- cells was
associated with cell proliferation. They theorize that the
CD34+CD38-- cells that proliferate in response to individ-
ual cytokines possibly represent a more mature progenitor
subpopulation than those that survive without undergo-
ing cell division. It is possible that this more mature sub-
population that proliferates in response to cytokines
could represent the CD34+CD38--c-mpl-- HSC/PC frac-
tion. Because the CD34+CD38-- subset represents a heter-
ogeneous population, assaying for c-mpl antigen density
using selective monoclonal antibodies such as CD110
(clone 3G4) [21,22] may offer a practical method to select
a more primitive HSC/PC within the subset from any tis-
sue source, potentially representing a high percentage of
the long-term multilineage repopulating capacity of
CD34+CD38-- HSC/PC [33]. Importantly, we found for all
tissues, that the intensity of c-mpl expression can define a
relatively homogeneous population of CD34+ MNC with
high CD34 and very low CD38 expression.

Studies of patients with congenital amegakaryocytic
thrombocytopenia (CAMT) point to the essential role of
c-mpl+ HSC subsets in maintaining human long-term
hematopoiesis in vivo. The pathophysiology of CAMT was


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I 7
I 10 102 10 ,7 1
- CD34-FITC


Journal of Translational Medicine 2006, 4:9


*3







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A
Injections

CD34'38./dimcmpI- u


Donor Cell Gate B

t


- FSC --- HLA-B8-APC
-1 0I,


C CD2+B8+Gate D

HL -B-AP 10



I1 1 2 1 V 1V
4- HLA-B8-APC

- -1 1t


- CD4-FITC -- HLA-B8-APC - HLA-B8-APC -


CD34*38-ldimc-mpl-


t2
No injection '
Control

i


-- FSC HLA-B8-APC 4- HLA-B8-APC - CD4-FITC -"- 4- HLA-B8-APC - HLA-B8-APC --

Figure 4
Human c-mpl+ HSC/PC have increased T-lineage repopulation capability compared with the corresponding c-
mpl-- HSC/PC. Small I mm3 fragments of human HLA-B8-- fetal thymus and fetal liver were co-implanted, adjacent to one
another, under the renal capsule of scidlscid (SCID) mice, generating one SCID-hu Thy/Liv graft per mouse. Viable, robust 8-
week old Thy/Liv grafts (n = 21) were exposed to sublethal irradiation (400 Rads) in vivo, then injected with either 60,000 or
30,000 sorted human HLA-B8+ CD34+CD38--/dimc-mpl+ or CD34+CD38--/dimc-mpl-- ABM cells and allowed to engraft for 8
weeks. Three grafts were reserved as non-injected controls. After 8 weeks, the Thy/Liv grafts were harvested, and single cell
suspensions prepared from these grafts were stained with PI and a panel of fluorescent-conjugated antibodies to T-lineage
markers and HLA-B8, with all appropriate isotype controls. 100,000 events from each graft were acquired on a FACSCaliburTM
flow cytometer. Representative examples of CD34+CD38--dimc-mpl+-injected grafts (n = 4), CD34+CD38--/dimc-mpl---injected
grafts (n = 7), and non-injected grafts (n = 3) are shown. A. Using FlowJo software, a pseudocolor dot plot of Forward Scatter
vs. Side Scatter was generated and a Human Lymphoid Cell Gate was applied to isolate engrafted human mononuclear cell
(MNC) events. B. Viable (PI--) human MNC events were then plotted on CD2-PE vs. HLA-B8-APC dot plot, and a CD2-PE+
gate was applied to isolate all viable CD2-PE+ MNC events. C. The total CD2-PE+ MNC events were next plotted on an HLA-
B8-APC histogram plot to apply HLA-B8-- and HLA-B8+ gates. The HLA-B8-- and HLA-B8+ gates were generated from the three
non-injected Thy/Liv grafts combined. The HLA-B8- gate was defined to include 99.0% of the left-most viable MNC events of
the non-injected grafts on an HLA-B8-APC log histogram plot. The HLA-B8+ gate was drawn from the end of the HLA-B8--gate
to the far right of the histogram plot. The percentage of viable (PI--) CD2-PE+ HLA-B8+ events, out of the total viable CD2-PE+
MNC population, is shown for the representative examples of the three treatment arms. D. CD4-FITC vs. CD8-PE pseudo-
color dot plot of viable (PI--) Human Lymphoid Cell gated events. Gates were applied to depict the thymocyte subsets
(CD4+CD8+, CD4+CD8-, CD4-CD8+). E. Total viable (PI--) CD4+CD8-- MNC events were plotted on an HLA-B8-APC log
histogram plot and the HLA-B8+ gate was applied. The percentage of viable donor-derived CD4+CD8--HLA-B8+ events out of
the total viable CD4+CD8-- MNC events is shown for the representative examples of the three treatment arms. F. Total viable
(PI--) CD4--CD8+ MNC events were plotted on an HLA-B8-APC log histogram plot, the HLA-B8+ was applied, and the percent-
age of viable, donor-derived CD4--CD8+HLA-B8+ MNC events out of the total viable CD4--CD8+ MNC population in the Thy/
Liv graft was calculated.


recently determined to be the result of mutations in the c-
mpl gene, leading to defective responsiveness to TPO [34].
Ballmaier et al. found that the percentage of CD34+ BM-
MNC in younger CAMT patients near their time of diagno-
sis was within the range of normal age-matched controls,
but progressively and consistently declined as patient age
increased [34,35]. Furthermore, the CD34+ BM-MNC
from CAMT patients showed a diminished potential to


form myeloid, erythroid and megakaryocytoid CFU in
vitro compared to normal controls, and this reduced
potential was accentuated with increasing age. Recently,
Koka et al. [36] reinforced our previous in vivo SCID-hu
Bone repopulation study [20] by finding that erythroid,
myeloid and megakaryocytoid in vitro colony-forming
activity (CFA) of secondarily engrafted human CD34+
HSC/PC exposed to human immunodeficiency virus type


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- FSC HLA-B88-APC - HLAB8B-APC t CD4-FITC HLA-B8-APC HLAB8B-APC -







http://www.translational-medicine.com/content/4/1/9


Table 4: Thymocyte Repopulation of SCID-hu Thy/Liv Grafts


HLA* Donor Cell Transplant


Non-injected Control


CD341CD38--4di-cmpl--


CD341CD38--4dincmplj


No. Mice Engrafted NA 4/7

% Donor CD2+ 2.66 2.80 (3.27)
% Donor CD4+8+ 0.82 0.48 (0.47)
% Donor CD4+8- 9.84 30.7 (28.9)
% Donor CD4-8+ 5.17 18.1 (14.3)

HLA, human leukocyte antigen; SCID-hu Thy/Liv, scidlscid mouse with human thymus/liver implants


1 (HIV-1) infection in primary SCID-hu Thy/Liv grafts
correlated with c-mpl expression. They also found that
secondary erythroid, myeloid and megakaryocytoid CFA


Viable CD2+ Cells


in vitro was inhibited by blocking antibodies to c-mpl
administered in vivo to human HSC/PC residing within
primary SCID-hu Thy/Liv grafts. Furthermore, a recent


= No Injection
- CD34+CD38-/dimc-mpl-
- CD34+CD38-/dimc-mpl+


Figure 5
Human c-mpl+ HSC/PC show significant repopulation of mature single positive CD4+ and CD8+ thymocytes.
SCID-hu Thy/Liv grafts (HLA-B8-; n = 21) were sublethally irradiated and injected with 60,000 or 30,000 sorted HLA-disparate
(HLA-B8+) CD34+CD38--dimc-mpl+ or CD34+CD38--/dimc-mpl- human ABM cells and allowed to repopulate for 8 weeks. Three
grafts were reserved as non-injected controls. The surviving Thy/Liv grafts (c-mpl' = 4; c-mpl-- = 7; Controls = 3) were har-
vested and single cell suspensions were stained with PI and fluorescence-conjugated antibodies against T-lineage markers, the
HLA-B8 donor marker, and appropriate isotype controls. Events (100,000) were acquired and analyzed using FlowJo software
to determine the percentage of viable (PI--) donor-derived (HLA-B8+) MNC expressing the different T-lineage markers from
among all viable MNC expressing that particular marker. Viable (PI--) MNC events from the non-injected Thy/Liv grafts were
analyzed to establish the HLA-B8--gate (representing 99.0% of the left-most events on an HLA-B8-APC log histogram plot) and
the HLA-B8+ gate (extending from the end of the HLA-B8-- gate to the far right of the log histogram). These gates were then
applied without alteration to the HLA-B8-APC log histogram plots of thymocyte subsets from the c-mpl+ and c-mpl---injected
Thy/Liv grafts. A. The mean percentage of viable (PI--) CD2-PE+ HLA-B8+ events, out of the total viable CD2-PE+ MNC popula-
tion is shown for the non-injected grafts (n = 3), the CD34+CD38--c-mpl---injected grafts (n = 7), and the CD34+CD38--c-mpl+-
injected grafts (n = 4). Error bars = SEM. B. The mean percentage of viable (PI--) HLA-B8+ donor-derived CD4/CD8 subset
thymocytes [CD4+CD8-, CD4--CD8+, CD4+CD8+, CD4--CD8--], out of the respective total thymocyte subset population, is
shown for the three treatment arms. Error bars = SEM.




Page 13 of 18
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6.83 (4.48)
0.92 (0.64)
54.1 (35.4)
29.2 (19.4)


CD4+CD8+ CD4+CD8- CD4-CD8+
Viable CD4 CD8 Subsets


Journal of Translational Medicine 2006, 4:9







http://www.translational-medicine.com/content/4/1/9


Table 5: One Way ANOVA of SCID-hu Thy/Liv Thymocyte Repopulation


Thymocyte Subset


One way ANOVA


Dunnett's Multiple Comparison Post-Test


Linear Trend Post-Test


c-mpl vs. control

P < 0.05
NS
P < 0.05
P <0.01


c-mpl-- vs. control

NS
NS
NS
NS


Values represent calculated p values for the indicated analyses. ANOVA, analysis of variance; NS, not significant (p > 0.05); SCID-hu Thy/Liv, scid/
scid mouse with human thymus/liver implants


report documents a patient with Systemic Sclerosis and an
autoantibody to c-mpl who developed pancytopenia, pro-
viding a naturally occurring example of a blocking anti-
body to c-mpl and its resultant inhibitory impact on
human multilineage hematopoiesis in vivo [37].

Studies have suggested the existence of HSC from select
tissue sources in early fetal life that are intrinsically differ-
ent [38-40] and more primitive than HSC isolated from
various tissue sources in later development and adult life
[41]. We found no significant differences in c-mpl expres-
sion between the different tissue sources of HSC. Several
authors have found a greater proportion of immature
(CD38-- and/or HLA-DR--) CD34+ cells in UCB compared
to the other tissue sources, in which no differences were
noted [42-51]. Sovalat et al. [42] found that UCB CD34+
cells had a significantly higher percentage of the
CD34+CD38- subset than did CD34+ cells from normal
ABM, mobilized ABM, or mPBSC. The percentage of
CD34+ HSC/PC from normal PB that were CD38--was sig-
nificantly lower than in CD34+ cells from all other tissue
sources. While we did not address this question directly,
our study examining differentiation stage subsets raises
the possibility that differences in stem cell capacity
between tissue sources may be explained by differences in
the quantity ofc-mplBright/+ HSC residing within the tissue,
rather than by qualitative differences in c-mpl antigen
expression of HSC.

Henon et al. [52,53] found that the CD34+CD38- HSC/
PC subset was fundamentally important for early and late
multilineage engraftment following autologous mPBSC
transplantation. They found no correlation with infused
numbers of MNC, CD34+CD33- cells or CD34+CD33+
cells, and weak, inconsistent correlation of CFU-GM,
CD34+ cells or CD34+CD38+ cells, with the number of
days to reach predetermined recovery parameters of abso-
lute neutrophil count, platelet count and reticulocyte
count. Although previous studies have defined a CD34+
cell threshold value above which reliable and rapid
engraftment was stated to occur actual demonstration of
a linear correlation between infused CD34+ cells and neu-


trophil engraftment was rarely reported [52]. Indeed, in
their study, the use of a CD34+ threshold value as a predic-
tive factor for engraftment was limited: in 24% (11/45) of
their patients, the CD34+ content was not predictive for
rapid hematopoietic recovery. The authors discovered,
rather, that the content of CD34+CD38- cells in the graft
products was the strongest and most reliable predictive
factor for both short-term and long-term tri-lineage
hematopoietic engraftment. Our study provides evidence
for the theory that this is explained mechanistically by the
high level of c-mpl+ expression within this stage I mPBSC
subset.

Henon and colleagues found that a threshold value of 5 x
104 CD34+CD38- cells/kg BW is a reliable tool to predict
engraftment [52]. Patients that received >5 x 104
CD34+CD38- cells/kg BW experienced a significantly
faster engraftment of neutrophils, platelets, and reticulo-
cytes than those that received less. Moreover, pre-trans-
plant total body irradiation conditioning regimens
significantly prolonged platelet and reticulocyte recovery
kinetics in only those patients whose CD34+CD38- cell
dose was <5 x 104 CD34+CD38- cells/kg BW. The authors
also found that post-transplant administration of hemat-
opoietic growth factors was beneficial for accelerating tri-
lineage engraftment in only those patients who received a
CD34+CD38- cell dose <5 x 104 cells/kg BW. Similarly,
post-transplant rh-G-CSF administration reduced the
length of hospitalization and post-transplant costs in only
those patients whose mPBSC infusion contained <5 x 104
CD34+CD3 8-- cells/kg BW [54]. In the setting of allogeneic
BMT for the treatment of leukemia or lymphoma, Waller
et al. [55] found that the CD34+CD38-- cell content of the
graft product was the best predictor for time to reach the
recovery parameters of absolute neutrophil count >1 x
109/L and platelet count >20 x 109/L. Based on our cur-
rent report and previous work [20], further studies are
warranted to determine whether the relevant cell type
within the CD34+CD38- subset identified by Henon,
Waller and colleagues as capable of rapid, sustained, tri-
lineage engraftment of both autologous mPBSC and allo-
geneic ABM transplants is the c-mplBright/+ cell.


Page 14 of 18
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CD2+
CD4+8+
CD4+8-
CD4-8+


P < 0.05
NS
P < 0.05
P < 0.05


P < 0.05
NS
P <0.001
P <0.001


Journal of Translational Medicine 2006, 4:9







Journal of Translational Medicine 2006, 4:9


Fox et al. [56-58] elucidated a critical role for TPO in
murine HSC engraftment following BMT. Five-fold more
murine ABM cells were needed to re-establish hematopoi-
esis following lethal irradiation in tpo-/- mice versus tpo+/+
mice. Thus, TPO appears to exert a short-term radiopro-
tective effect, presumably by enhancing the survival of the
c-mpl+ (and based on our study, predominantly
CD34+CD38--) HSC/PC residing in the tissue. This short-
term radioprotective effect ofTPO on its ligand-expressing
HSC/PC may explain the negative impact of total body
irradiation on patients who received very low doses of
CD34+CD38- cells in their mPBSC transplants, as
observed by Henon and colleagues [52]. Furthermore,
there was an in vivo expansion of 10-20-fold more long-
term repopulating HSC in the primary tpo+/+ recipients
versus the primary tpo--/- recipients. These results suggest
that the murine ABM c-mpl+ HSC subset contains HSC
that provide both short-term radioprotection and long-
term repopulation following BMT. Studies in mice have
also shown that in vivo [59] or ex vivo [60] priming of
donor ABM cells with TPO can accelerate the reconstitu-
tion of platelets and red cells and ameliorate post-trans-
plant thrombocytopenia.

Murine TPO mRNA is expressed by RT-PCR in embryonic
stem cells, whereas murine c-mpl mRNA is expressed in
the embryonic yolk sac, at day 3 of embryoid body in vitro
differentiation, and in the blast-colony forming cell, an in
vitro surrogate for the hemangioblast common precursor
to the endothelial and hematopoietic lineages [61]. These
findings indicate a potential role for the TPO/c-mpl pair
in maintaining the self-renewal potential of the heman-
gioblast. It will be important to determine whether c-mpl
is expressed in the recently identified murine embryo
primitive streak-derived brachyury+ Flk-1+ cell population,
within which the hemangioblast resides [62].

Conclusion
This report demonstrates that human c-mpl surface recep-
tor is expressed at the highest density on the most primi-
tive subset within the human CD34+ HSC/PC population,
regardless of the tissue source or stage in ontogeny. In
addition, these c-mpl+ HSC/PC can also provide for
thymic reconstitution and T-cell lineage repopulation in a
manner that is at least as robust as, if not more robust
than, that of the c-mpl-- progenitor subset. Our results
reported here, combined with our previous studies of c-
mpl+ versus c-mpl- competitive HSC repopulation in
murine and human model systems [20], building on the
investigations of TPO by others [11,12,33,63], suggest
that c-mpl is a selective, practical and physiologically rel-
evant marker of human long-term multilineage repopu-
lating HSC. It has advantages in being more selective than
CD34, its function relevant to HSC biology is more fully
characterized than candidate markers such as CD133 [64-


http://www.translational-medicine.com/content/4/1/9



67] or CDCP1 [7,8], and isolation strategies may be less
toxic and technologically simpler than DNA binding dyes
to identify side population cells [68], or fluorescent sub-
strates to identify aldehyde dehydrogenaseHi cells [69,70].
Studies are indicated to evaluate whether c-mplBright/+ cell
number may be a superior measure for monitoring mobi-
lization levels of peripheral blood for the timing and opti-
mization of apheresis collections, and a more reliable and
predictive gauge of stem cell content prior to transplant or
following expansion. Positive selection protocols using
monoclonal antibodies to c-mpl could also prove to be
useful and efficient purification methodologies for stem
cell banking and solid tumor purging or T-cell depletion
of stem cell products.

List of abbreviations
7-AAD, 7-amino-actinomycin D; ABM, adult bone mar-
row; AgD, antigen density; ANOVA, analysis of variance;
APC, allophycocyanin; BM, bone marrow; BMT, bone
marrow transplantation; BW, body weight; CAMT, con-
genital amegakaryocytic thrombocytopenia; CD, cluster
of differentiation; CFA, colony-forming activity; CFC, col-
ony-forming cells; CFU, colony-forming units; FITC, fluo-
rescein isothyocyanate; FL, fetal liver; FSC, forward scatter;
G-CSF, granulocyte-colony stimulating factor; HIV-1,
human immunodeficiency virus type 1; HLA, human leu-
kocyte antigen; HSC, hematopoietic stem cell(s); HSC/
PC, hematopoietic stem/progenitor cell(s); LTC-IC, long
term culture-initiating cell(s); MB, MACS Buffer; MFI,
median fluorescence intensity; MNC, mononuclear cells;
NC, nucleated cells; NOD, non-obese diabetic; PB,
peripheral blood; mPBSC, cytokine-mobilized peripheral
blood stem cells; PBS, phosphate-buffered saline; PE, phy-
coerythrin; PI, propidium iodide; QIIF, quantitative indi-
rect immunofluorescence; RMFI, Relative MFI; RT-PCR,
reverse transcriptase-polymerase chain reaction; SCID,
severe combined immunodeficiency (scid/scid) mouse;
SCID-hu Thy/Liv, scid/scid mouse with human thymus/
liver implants; SD, standard deviation; SED, super-
enhanced Dmax subtraction; SEM, standard error of
mean; SM, staining medium; SP, side population; SSC,
side scatter; TPO, thrombopoietin; UCB, umbilical cord
blood.

Competing interests
WGK has previously served as a consultant to Genentech,
Inc. and currently receives funding from Genentech (co-
developer ofTPO) for the identification of novel antibody
therapeutics in hematologic malignancies.

Authors' contributions
WGK and JMN designed the study. WGK established the
SCID-hu mouse colony and provided the necessary equip-
ment and reagents. LCJ and CRC contributed to the pro-
curement and collection of specimens. JMN performed


Page 15 of 18
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Journal of Translational Medicine 2006, 4:9


the experiments, acquired the data, analyzed and inter-
preted the results, and drafted and revised the manuscript.
WGK, CRC and LCJ reviewed, critiqued and contributed
to the final manuscript.


Acknowledgements
We are indebted to Dr. Dan Eaton and Genentech, Inc. for the use of the
3G4 anti-c-mpl monoclonal antibody. We thank Mary Albertus, Robert
Sachs, and Mark Wall of the Hospital of the University of Pennsylvania
Pathology Department for assistance with the UCB and mPBSC collections.
We acknowledge the capable technical assistance of Heather Mclntosh in
the SCID-hu Thy/Liv reconstitution studies. We thank Hank Pletcher,
Andrew Morschauser, Nikki Brake and Dr. Jonni Moore of the University
of Pennsylvania Cancer Center Flow Cytometry Facility for their excellent
technical assistance with the cell sorts. We acknowledge Jodi Kroeger and
Johana Melendez of the Flow Cytometry Core Facility, Larry Kuba and Jen-
nifer Hockenbury of the Molecular Imaging Core Facility, and Joan Miller
and Sue Felber of the Medical Library, at the H. Lee Moffitt Cancer Center
and Research Institute. We appreciate the assistance of Susan Arab and
Patricia Massard with manuscript preparation, and we thank Dr. Kaaron
Benson for reviewing the manuscript. The first author sincerely appreciates
the helpful discussions provided by Dr. Tomar Ghansah, Caroline Desponts
and Dr. Jia-Wang Wang during the course of this research. WGK was sup-
ported by grants from the National Institutes of Health (RO I DK 54767,
R21 A144333, POI NS27405 and RO I HL72523), the Penn Research Foun-
dation, and academic development funds from the H. Lee Moffitt Cancer
Center & Research Institute and the University of South Florida. JMN was
supported by training grants from the National Heart, Lung and Blood Insti-
tute (T32 HL07775) and the National Institute of Allergy and Infectious Dis-
eases (T32 CA09140). WGK is the Newman Family Scholar of the
Leukemia and Lymphoma Society. CRC was supported by a Shands Hospi-
tal Research and Development Grant.

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a small animal model for HIV infection and pathogenesis.
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