|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2813-2820
HEMATOPOIESIS
Isolation and characterization of human
CD34 Lin and
CD34+Lin hematopoietic stem cells using
cell surface markers AC133 and CD7
Lisa Gallacher,
Barbara Murdoch,
Dongmei M. Wu,
Francis N. Karanu,
Mike Keeney, and
Mickie Bhatia
From The John P. Robarts Research Institute, London,
Ontario, Canada, and the Department of Microbiology and Immunology,
University of Western Ontario, London, Ontario, Canada.
 |
Abstract |
Recent evidence indicates that human hematopoietic stem cell
properties can be found among cells lacking CD34 and lineage commitment markers (CD34 Lin). A
major barrier in the further characterization of human
CD34 stem cells is the inability to detect this
population using in vitro assays because these cells only
demonstrate hematopoietic activity in vivo. Using cell surface
markers AC133 and CD7, subfractions were isolated within
CD34CD38Lin and
CD34+CD38Lin cells derived
from human cord blood. Although the majority of CD34CD38Lin cells
lack AC133 and express CD7, an extremely rare population of
AC133+CD7 cells was identified at a
frequency of 0.2%. Surprisingly, these AC133+CD7 cells were highly enriched for
progenitor activity at a frequency equivalent to purified fractions of
CD34+ stem cells, and they were the only subset among the
CD34CD38Lin population
capable of giving rise to CD34+ cells in defined liquid
cultures. Human cells were detected in the bone marrow of
non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks
after transplantation of ex vivo-cultured AC133+CD7 cells isolated from the
CD34CD38Lin population,
whereas 400-fold greater numbers of the
AC133CD7 subset had no engraftment
ability. These studies provide novel insights into the hierarchical
relationship of the human stem cell compartment by identifying a rare
population of primitive human CD34 cells that are
detectable after transplantation in vivo, enriched for in vitro
clonogenic capacity, and capable of differentiation into
CD34+ cells.
(Blood. 2000;95:2813-2820)
© 2000 by The American Society of Hematology.
| |
Introduction |
The human hematopoietic system is sustained by rare
pluripotent stem cells that are capable of extensive proliferation and differentiation.1 Utility of human stem cells ranges from
gene therapy for the correction of genetic disorders to ex vivo
expansion and transplantation for the purposes of hematological
recovery and long-term engraftment in patients undergoing aggressive
cancer therapy.2-4 Evidence in mice, monkeys, and humans
indicates that the most primitive blood stem cells reside in a
phenotypically and functionally heterogeneous population of cells
having "stem cell" attributes.5-8 Therefore the apex
of the hematopoietic hierarchy appears to comprise an array of
primitive cells that form the stem cell compartment. The bulk of
experimental studies to support this notion comes from purification of
various primitive subsets using cell surface markers or DNA
stains.9-12 Subpopulations derived from hematopoietic
sources can be isolated and subjected to in vitro and in vivo assays
that are capable of measuring stem cell function.12,13 The
most critical marker used for the isolation of human blood stem cells
is the sialomucin CD34, and accordingly, all experimental
and clinical protocols involving gene transfer, stem cell
transplantation, and expansion are designed for CD34+ subsets.
Previous studies in the murine system indicate that some stem cells
capable of long-term repopulation do not express detectable levels of
cell surface CD34.10 A study by Osawa et al12
demonstrated that single murine
CD34Lin cells could be
transplanted into lethally irradiated mice and sustain long-term
multilineage engraftment, but they were devoid of short-term
repopulation ability. Recently primitive populations of cells with DNA
dye efflux properties ("SP cells") have been isolated from monkey
and human hematopoietic tissue.14 These cells do not
express cell surface CD34. While monkey-derived SP cells were shown to
possess primitive hematopoietic capacity using in vitro assays, human
CD34Lin cells were unable to
demonstrate hematopoietic activity in vitro,14 and it remained unclear whether the human
CD34CD38Lin
cells had any primitive hematopoietic function. Using in vivo transplantation assays, the groups of Zanjani15 and
Dick16 demonstrated that a population of
CD34Lin cells was capable of
engrafting non-obese/severe combined immunodeficiency (NOD/SCID) mice
and fetal sheep. These studies provided the first and only evidence of
hematopoietic activity from
CD34Lin cells derived from human
tissue. From these earlier studies, it has been shown that the
CD34Lin population is made up of
a large number of cells found in human cord blood (CB) and bone marrow
(BM). CD34Lin stem cells have
been hypothesized to be the most primitive blood cells identified to
date, and it is surprising that this population comprises a 3- to
4-fold greater number of cells than all other CD34+
subfractions combined. However, the frequency of repopulating cells
within the CD34Lin fraction was
shown to be far lower than that found in the CD34+
subfraction.16,17 Therefore, the biological function of
CD34 stem cells more closely resembles the rare
frequency expected of such a primitive cell and may suggest the absence
of a sufficiently purified population of human stem cells devoid of CD34.
Using cell surface markers CD7 and AC133,18-20 we
identified biologically distinct subsets from human
CD34CD38Lin
and CD34+CD38Lin
populations. A unique subset expressing AC133 and lacking CD7 was found
at a frequency of 0.2% within the
CD34CD38Lin
fraction, which contained all of the progenitor capacity
previously thought to be deficient in human CD34
stem cells.14,16 Furthermore, using defined in vitro
culture conditions, these
AC133+CD34CD38Lin
cells were capable of acquiring CD34 and possessed a clonogenic progenitor capacity equivalent to primitive CD34+ cells.
Human cells were detected in the BM of NOD/SCID mice after transplantation of ex vivo-cultured
AC133+CD34CD38Lin
cells, which suggests that this population contains primitive repopulating cells. Identification of these cells demonstrates a
previously uncharacterized heterogeneity within the human
CD34Lin population and provides
insights into the relationship of CD34 cells to
other cells in the human stem cell compartment.
| |
Materials and methods |
Human cells
Samples of human CB were obtained from placental and umbilical
tissues and diluted 1:3 in Iscove's modified Dulbecco's medium (IMDM)
or  -modified Eagle medium (-MEM) (Gibco Life
Technologies, Grand Island, NY). The mononuclear cells
were collected by centrifugation (Ficoll-Hypaque, Pharmacia Biotech,
Uppsala, Sweden).
Cell purification
AC133 and CD7 subsets were isolated and analyzed from
CD34CD38Lin
and CD34+CD38Lin
cells using standard protocols.16 CB cells were first
enriched for lineage-depleted (Lin) cells by
negative selection using a cocktail of lineage antibodies and a device
similar to that described by the manufacturers (StemSep; Stem Cell Technologies Inc, Vancouver, BC, Canada). These cell fractions were then stained with antihuman
CD38 allophycocyanin (APC), antihuman CD34
peridinin chlorophyll protein (Per-CP), antihuman CD7 fluorescein
isothiocyanate (FITC) (Becton Dickinson Immunocytometry Systems, San
Jose, CA), and anti-hu AC133 phycoerythrin (PE) (Miltenyi Biotech,
Bergisch Gladbach, Germany) and then analyzed and sorted on a
fluorescence-activated cell sorter (FACS) (FACS Vantage SE, Becton
Dickinson). Sorting gates used are indicated in Figure
1. Data acquisition and analysis were then
performed (Cell Quest software, Becton Dickinson).
View larger version (37K):
[in this window]
[in a new window]
| Fig 1.
Phenotypic analysis of cell surface
AC133 and CD7 on primitive
subpopulations of
CD34CD38Lin and
CD34+CD38Lin
populations of human CB cells.
Representative analysis of cell surface CD34 and CD38 by multiparameter
flow cytometry of human CB cells depleted for cells expressing lineage
commitment markers (Lin). (A) Subpopulations of
CD34CD38Lin
and CD34+CD38Lin
cells were gated R1 and R2, respectively. Histograms showing the
expression of CD7 and AC133 on gated subfractions of (B, C)
CD34CD38Lin
cells and (D, E)
CD34+CD38Lin cells.
Due to the low number of AC133 cells detected in the
CD34CD38Lin
fraction, cells shown in (C) are also gated CD7
using (B) markers. (A to E) Markers indicate sorting gates used for the
isolation of specified subfractions. Analysis is representative of 4 independent CB samples. A single-line histogram indicates isotype
overlay.
|
|
Clonogenic progenitor assays
Human clonogenic progenitor assays were performed by plating various
sorted cell populations at concentrations ranging from 1 × 102 cells to upward of
1 × 103 cells into a methylcellulose cocktail
(MethoCult H4434, Stem Cell Technologies) containing 50 ng/mL
recombinant human (rH) stem cell factor (SCF), 10 ng/mL rH
granulocyte-macrophage-colony-stimulating factor (GM-CSF), 10 ng/mL rH
interleukin-3 (IL-3), and 3 units/mL rH erythropoietin. The cocktail
was then incubated at 37°C with 5% carbon dioxide
(CO2) in a humidified atmosphere. Differential colony
counts were scored after 10-14 days by morphological characteristics using an inverted microscope.
Liquid suspension cultures
Sorted cells were incubated in serum-free conditioned BSA, insulin,
transferrin (BIT) media (Stem Cell Technologies) that was
previously shown to maintain primitive human populations.21 Briefly, the serum-free BIT media was supplemented with
104 mol/L  -mercaptoethanol (2 mmol/L),
L-glutamine, and human growth factors. The growth factor cocktail was
used at final concentrations of 300 ng/mL of SCF (Amgen Inc, Thousand
Oaks, CA) and Flt-3 (R & D Systems, Minneapolis, MN); 50 ng/mL
G-CSF (R&D Systems); and 10 ng/mL of IL-3 and IL-6 (R & D
Systems). Cells were cultured in flat- bottomed suspension wells of
96-well plates (Falcon, San Jose, CA) and incubated the
indicated times at 37°C and 5% CO2 with 50 µL fresh
media. The growth factor cocktail was added to each well every other day.
Transplantation of purified cells into NOD/SCID mice
Cells were transplanted by tail vein injection into sublethally
irradiated NOD/LtSz-scid/scid (NOD/SCID) mice (350 rads137 cesium) according to standard
protocols.16 The mice were sacrificed 6-8 weeks after
transplantation in accordance to local animal welfare protocols, and BM
cells were collected from femurs, tibiae, and iliac crests.
Analysis of human cell engraftment
High molecular weight DNA was extracted from BM cells of
transplanted mice and digested with EcoR1 restriction enzyme (MBI Fermentus, Flamborough, Ontario, Canada). The percentage
of human cells was determined by probing Southern blot analyses with a human chromosome 17-specific -satellite probe as previously
described.16 The level of human cell engraftment was
determined by analysis of Southern blots (PhosphoImager; Molecular
Dynamics, Sunnyvale, CA) and quantified by using software (Image-Quant;
Molecular Dynamic, Sunnyvale, CA) to compare the characteristic
2.7-kilobase (2.7-kb) band with human:mouse DNA mixture controls.
This was accomplished by using a lower limit of detection
of 0.05% human DNA, which provided a linear signal response. In cases
where the level of human cells was less than or equal to 0.05%,
polymerase chain reaction (PCR) for the human-specific gene
CART-1 was used as shown previously.22 Briefly,
CART-1 primers 5'-AAGGATACCACAATAAGCTGC-3' and
5'-GGTTTGTGGAGACTGGCAC-3' were used to amplify
(Perkin-Elmer 9700; Perkin Elmer, Norwalk, CT) a 156-base pair
(156-bp) product from the untranslated region of the human
CART-1 gene at 96°C for 2 minutes followed by 35 cycles at
94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 15 seconds at 1 mmol/L magnesium dichloride (MgCl2). In
addition, the BM of transplanted mice was analyzed by staining with the
human pan-leukocyte marker CD45 to detect the presence of human
hematopoietic cells by flow cytometric analysis using a FACS
(FACSCalibur, Becton Dickinson) as described below.
Flow cytometric analysis of murine BM
To prepare cells for flow cytometry, contaminating red cells were
lysed with a 0.8% ammonium chloride solution, and the remaining cells
were washed in phosphate-buffered saline (PBS) containing 5% fetal
calf serum (FCS). Approximately 106 cells were resuspended
in 1 mL PBS and 5% FCS, washed, and then incubated with monoclonal
antibodies (mAbs) at a concentration of 5 µg/mL for 30 minutes at
4°C. The mAb combinations used are indicated in Figure 5B. (CD45
was conjugated to PerCP; CD20 and CD33 were conjugated to FITC; CD38,
CD15, and CD19 were conjugated to PE; CD34 was conjugated to APC.)
Cells were then washed 3 times in PBS plus 5% FCS and analyzed
(FACSCalibur or FACS Vantage SE, Becton Dickinson). For each mouse
analyzed, an aliquot of cells was also stained with mouse
IgG1 conjugated to FITC, PE, PerCP, and APC as an isotype control.
| |
Results |
Phenotypic identification and isolation of subfractions comprising
CD34CD38Lin and
CD34+CD38Lin populations
Mononuclear cells from human CB were enriched for cells that do not
express lineage-specific antigens (Lin) by magnetic
depletion of lineage-committed cells stained with a cocktail of mAbs
directed against a variety of lymphoid, myeloid, and erythroid
antigens.17 Figure 1A shows the analysis of CD34 and CD38
cell surface antigens expressed on a representative
Lin population measured by flow cytometry.
Subpopulations of interest were gated R1 and R2 for the analysis
of CD34CD38Lin
and CD34+CD38Lin
cells, respectively. Approximately 96% of the
CD34CD38Lin
population (gated R1), previously shown to contain no progenitor cell
activity in vitro but capable of modest repopulation in NOD/SCID mice,
was found to express cell surface CD7 (Figure 1B, Table 1). Although most of the
CD34CD38Lin
cells did not express AC133, a very rare population of cells within the
CD34CD38Lin
fraction that did not express CD7 were found to be AC133+
(Figure 1C).
View this table:
[in this window]
[in a new window]
|
Table 1.
Frequency of subpopulations within
CD34CD38Lin and
CD34+CD38Lin cells based on
cell surface AC133 and CD7 expression
|
|
CD34+CD38Lin cells
have been shown to be highly enriched for colony-forming cells
(CFCs); contain SCID repopulating cells (SRCs), which are capable of
multilineage repopulation in NOD/SCID mice; and comprise only 5%-8%
of the total CD34+ population.21,23 A low
frequency of
CD34+CD38Lin cells
(gated R2) were found to express CD7 on the cell surface (Figure 1D,
Table 1), whereas analysis of AC133 expression demonstrated 2 distinct
populations of AC133+ and AC133 cells
(Figure 1E). AC133+ and AC133 cells
represent previously unidentified subfractions within the highly
purified population of
CD34CD38Lin
and CD34+CD38Lin
cells that have yet to be characterized in functional assays. A summary
of the relative content and frequency of both AC133 and CD7
subpopulations within both
CD34CD38Lin
and CD34+CD38Lin
cells are shown in Table 1. AC133 and CD7 are therefore capable of distinguishing previously unknown phenotypic heterogeneity within
human subpopulations of both
CD34CD38Lin
and CD34+CD38Lin cells.
Clonogenic progenitor capacity of
CD34CD38Lin and
CD34+CD38Lin subpopulations
Hematopoietic assays that detect CFCs were used as a measure of
primitive progenitor cell function. Novel subpopulations identified within both
CD34CD38Lin
and CD34+CD38Lin
fractions were isolated based on the absence or presence of detectable cell surface CD7 or AC133 expression using sorting gates as
indicated in Figure 1. Sorted populations were reanalyzed to
assess purity, which was found to be greater than 98% (data not
shown). The CD7+ and AC133 subfractions
isolated from
CD34CD38Lin
cells had little to no detectable progenitor capacity (Figure 2A), which was similar to previous studies
using the
CD34CD38Lin
fraction purified from human sources of hematopoietic
tissue.14,16 It is important to note that more than 99% of
the cells within the
CD34CD38Lin
fraction are AC133CD7+ (Table 1).
CD34CD38Lin
cells that lack CD7 cell surface expression were capable of producing all types of CFCs at frequencies ranging from 40-60 colonies per 1000 cells (Figure 2A, Table 2). The frequency
of colony types from all subpopulations assayed for progenitor content
in Figure 2A and B are shown in Table 2. The population of even rarer
AC133+ cells within the
CD34CD38Lin
fraction (Table 1) could be isolated for functional analysis, but only
30-800 cells could be obtained from as much as 75 mL of whole cord
blood.
View larger version (22K):
[in this window]
[in a new window]
| Fig 2.
Clonogenic progenitor cell capacity of subfractions
isolated from
CD34CD38Lin and
CD34+CD38Lin
populations. CFC capacity was assessed in subfractions isolated
from (A)
CD34CD38Lin
cells and (B)
CD34V+CD38Lin cells
based on the absence or presence of CD7 and AC133 and represented as
the number of CFCs per 1000 purified cells. Sorting gates used for
isolation of subfractions are indicated in Figure 1 (A to E). Values
are the mean and the SEM of determinations from up to 4 separate CB
samples. Plating efficiencies were calculated by dividing the number of
cells plated by the number of CFCs detected.
|
|
Despite the low frequency of
AC133+CD34CD38Lin
cells, this subset demonstrated CFC capacity equal to that of
CD34+CD38Lin cells
(Figure 2A and B). As many as 400 CFCs were detectable per 1000 AC133+CD34CD38Lin
cells, thereby providing evidence for the identification of a rare cell
population that is functionally distinct from the remaining CD34CD38Lin
cells. Previous studies indicate that isolation of
CD34+CD38Lin cells
allow for the enrichment of CFCs to a frequency of 1 in 4.17,21 All subpopulations within the
CD34+CD38Lin
population demonstrated CFC capacity of multiple myeloid and erythroid
lineages, although cells expressing CD7 showed a lower frequency of
progenitors (Figure 2B, Table 2).
CD34+CD38Lin cells
lacking CD7 expression were able to generate progenitors at levels
previously demonstrated by the
CD34+CD38Lin
fraction.21 Isolated subpopulations of either
AC133 or AC133+ within the
CD34+CD38Lin fraction
(Figure 1E) were found to have equivalent CFC capacity (Figure 2B).
There was no significant difference in the type of CFCs produced
between these subpopulations, with one exception: in erythroid
progenitors, AC133 allows for discrimination of this lineage in the
AC133 subset (Table 2). With the exception of the CD7+
subfraction, all other subsets of the
CD34+CD38Lin
population had similar CFC capacity. Consistent with recent studies, all subsets within the
CD34CD38Lin
population displayed poor progenitor capacity in vitro, with the
exception of the
CD34CD38Lin
cells, which express AC133.
AC133+CD34CD38Lin
cells therefore represent a unique population of human
CD34 cells, which are enriched for primitive
hematopoietic activity.
Developmental capacity of subsets isolated from the
CD34CD38Lin population
We further characterized the developmental capacity of AC133 and CD7
subsets isolated from the
CD34CD38Lin
fraction. Using defined in vitro culture conditions previously shown to
support CD34+CD38Lin
cells,21 we cultured subsets of AC133+ and
AC133 cells and CD7+ and
CD7 cells for 3 days in these serum-free conditions.
Cell surface phenotype and side scatter properties of subpopulations
prior to culture are indicated as Day 0 (inset with isotype control, Figure 3). Responsiveness to serum-free
culture conditions was also assessed by visual inspection and defined
by changes in cell number or side scatter properties. CD7+
cells, which represent the majority of this fraction, remained unresponsive and showed no signs of proliferation (Figure 3A). In
contrast, the CD7 subfraction was capable of both
acquiring CD34 and demonstrating mild proliferation in response to in
vitro culture (Figure 3A). Similar to CD7+ cells,
AC133 cells derived from the
CD34CD38Lin
population did not respond to in vitro culture and were not capable of
acquiring CD34 surface expression (Figure 3B). However,
AC133+CD34CD38Lin
cells possessed the capacity to acquire cell-surface CD34 in vitro
(Figure 3B) and demonstrated a proliferative response by increasing the
total cell number by 3- to 4-fold after 3 days (data not shown). These
experiments suggest that the developmental capacity of
AC133+CD34CD38Lin
cells is biologically unique within the
CD34CD38Lin
population and contains the primitive precursors capable of
differentiation into CD34+ cells in vitro.
View larger version (35K):
[in this window]
[in a new window]
| Fig 3.
Analysis of CD34
expression of purified subpopulations
after in vitro culture for 3 days. A
representative experiment (n = 5) of CD34 cell surface expression
performed on subpopulations of cells isolated from the
CD34CD38Lin
population based on the absence or presence of (A) CD7 or (B) AC133
cell surface expression. The entire contents of individual wells was
collected after 3 days (2000-10 000 cells), washed, stained with mAbs
against CD34, and analyzed using flow cytometric analysis. Cell surface
phenotypes of subpopulations prior to culture are indicated as Day 0 (A, B) within isotype control dot plots.
|
|
Transplantation of AC133+ and AC133
cells from both
CD34CD38Lin and
CD34+CD38Lin subsets
into NOD/SCID mice
Primitive cells (SRCs) capable of repopulating NOD/SCID mice have
previously been shown to be enriched in the highly purified population
of CD34CD38Lin
and CD34+CD38Lin
cells from both human CB and BM, termed CD34SRCs and
CD34+SRCs, respectively.17,21,23 Other stem
cell-associated markers, such as Thy-1, human leukocyte
antigen-DR (HLA-DR), and c-kit, have been used for the selection of
subpopulations to further characterize and isolate primitive cells
within these fractions. But the markers are unable to identify
functionally distinct subsets within these
fractions.8,24,25 AC133+ and
AC133 cells were isolated from the
CD34CD38Lin
population and cultured in defined serum-free media previously shown to
enhance repopulating capacity in NOD/SCID mice. The level of human cell
engraftment was determined by analysis of DNA extracted from the BM
cells of recipient mice 8 weeks after intravenous transplantation of
cell fractions using PCR analysis for the human-specific gene sequence
CART-1.
Figure 4A shows the results of PCR using
human-mouse mixtures of DNA as controls. Indicated amounts of human DNA
were added to PCR reactions containing 200 ng of genomic DNA extracted
from the BM of nontransplanted NOD/SCID mice. This technique allowed for a linear signal at lower levels of detection compared with Southern
blot analysis used previously, which detected 0.05%
human cells, thereby extending the lower limit of detection for human cells (Figure 4A). As noted previously (Table 1), the proportion of
AC133+ cells within the
CD34CD38Lin
fraction is relatively low, and a range of only 30-800 cells could be
isolated from a single CB sample. Accordingly, in some cases individual
CB samples were pooled to obtain a greater number of cells, and
the average number of attainable cells can be estimated by multiplying
the range of
AC133+CD34CD38Lin
cells that can be isolated from a single CB sample (30-800 cells) by
the total number of samples pooled. Individual and pooled human CB
samples of 50-200 mL were used to purify AC133+ and
AC133 subsets from the
CD34CD38Lin
population, and the total cells attainable were either transplanted immediately after isolation (experiments 1 to 3, Figure 4B) or cultured in vitro for 3 days (experiments 4 to 8, Figure 4B) prior to
intravenous transplantation into NOD/SCID mice.
View larger version (47K):
[in this window]
[in a new window]
| Fig 4.
Transplantation into
NOD/SCID mice of ex vivo
cultured subsets of AC133+
and AC133 cells isolated from the
CD34CD38Lin
population. (A) Human-mouse DNA mixtures were used for PCR
amplification of CART-1 human-specific gene sequence. A total
of 200 ng of genomic DNA from BM cells of nontransplanted control
NOD/SCID mice was mixed with equal volumes of serially diluted (0, 0.0625, 0.125, 0.25, and 0.5 ng) human genomic DNA. There were no
detectable PCR products in the absence of human DNA, indicating the
specificity of the PCR reaction to human sequence. However, increased
amounts of human DNA allowed for the detection of a linearly increasing
signal. PCR reactions of the human-mouse DNA mixture were compared to
200 ng DNA extracted from NOD/SCID mice transplanted with ex
vivo-cultured cell populations as indicated. (B) Summary of the level
of human cell engraftment detectable in NOD/SCID mice transplanted with
AC133+ and AC133 subfractions isolated
from the
CD34CD38Lin
populations in 8 independent experiments. Experiments 1 to 3 represent
transplantation of NOD/SCID with uncultured de novo isolated
subfractions, whereas experiments 4-8 represent results from mice
transplanted with subfractions cultured for 3 days in serum-free media.
Experiments 2, 3, and 8 represent transplantation of cells isolated
from 3 pooled CB samples.
|
|
In 3 experiments, de novo isolation of AC133+ or
AC133 subsets demonstrated that only 1 out of 3 NOD/SCID recipient mice contained human cells when transplanted with
the AC133+ subfraction, whereas there were no human cells
detected in animals transplanted with the AC133
cells (Figure 4B). However, after ex vivo culture, as few
as 30 AC133+CD34CD38Lin
human cells were detected in the BM of 4 out of 6 NOD/SCID mice, whereas 44 000 and 150 000
AC133CD34CD38Lin
transplanted cells were not detectable (Figure 4A and B). A
representative analysis of 3 experiments is shown adjacent to
human-mouse mixtures (Figure 4A) for mice transplanted with cultured
AC133 and AC133+ subfractions.
Transplantation of 5 × 106
CD34Lin+ control carrier cells was not
detectable in the BM of recipient mice (Figure 4A). Based on an average
of 80 × 106 cells comprising the total murine BM
and molecular weight of human genomic DNA per cell, it can be estimated
that our level of detection represents an average of 15 000 human
cells in the BM of transplanted mice. This estimate suggests that
AC133+CD34CD38Lin
cells are capable of a significant level of cellular expansion and
survival over an 8-week period in vivo as compared with 400 times the
number of transplanted
AC133CD34CD38Lin
cells that were not detectable. Taken together, these experiments demonstrate that rare AC133+ cells possess in vivo capacity
which is distinct from the bulk population of
CD34CD38Lin cells.
For comparison, AC133+ and AC133 cells
and CD7+ and CD7 cells were isolated
from the CD34+CD38Lin
population and transplanted into NOD/SCID mice to evaluate their repopulating capacity. Figure 5A summarizes
the levels of human cell engraftment and frequency of SRC detection
within the indicated subfractions from 4 independent CB samples.
Engraftment was detected after transplantation with as few as 900 CD34+CD38Lin
cells expressing AC133. With the exception of 1 animal transplanted with the highest dose of
AC133CD34+CD38Lin
cells (20 000 cells), this novel population was devoid of SRC activity. In addition, the CD7 subpopulation
contained SRCs, whereas the CD7+ population did not contain
repopulating cells (Figure 5A). Because all AC133+ cells
are CD7 in this fraction, the
AC133+CD7 cells represent a unique
population within the
CD34+CD38Lin fraction
that is enriched for primitive repopulating cells.
View larger version (26K):
[in this window]
[in a new window]
| Fig 5.
Analysis of SRC capacity in subpopulations of
CD34+CD38Lin cells
based on the expression or
absence of cell surface CD7
and AC133. (A) Summary of the level of human
engraftment in NOD/SCID mice transplanted with AC133+ and
AC133 subfractions and CD7+ and
CD7 subfractions isolated from the
CD34+CD38Lin
population of human CB. Eight weeks after transplantation, the presence
of human cells in the BM of 22 mice was assessed by extraction and
hybridization of DNA using either Southern blot analysis with a human
chromosome 17-specific -satellite probe or multiparameter flow
cytometric analysis with the human-specific pan-leukocyte marker
CD45. Each symbol represents a single NOD/SCID recipient. (B)
Multilineage differentiation of human
AC133+ + CD34+ + CD38Lin
cells in NOD/SCID mice. Bone marrow from a representative engrafted
mouse transplanted with 10 000
AC133+ + CD34+ + CD38Lin
CB cells was stained with various human-specific mAbs and
analyzed by flow cytometry. (I) Histogram of CD45 (pan-leukocyte
marker) expression indicates that 65% of the cells present in the
murine BM were human. Analysis of lineage markers was done on cells
within gate R2 (CD45+). Histogram overlay (single line)
represents isotype control for nonspecific IgG staining. Expressions of
(II) myeloid marker CD33 and mature myeloid marker CD15, (III) pan-B
cell markers CD19 and CD20, and (IV) CD38 and immature hematopoietic
marker CD34 are shown. Multilineage engraftment shown here was similar
to that found in mice transplanted with
CD7CD34+CD38Lin
cells (data not shown).
|
|
The differentiative and proliferative capacity of highly purified SRCs
from CD34+CD38Lin
cells selected for AC133 expression was assessed by flow cytometric analysis of engrafted NOD/SCID mice 8 weeks after transplantation. A
representative analysis of a NOD/SCID mouse transplanted with 10 000
AC133+CD34+CD38Lin
cells is shown in Figure 5B. The BM of this mouse contained 65% CD45+ human cells (Figure 5B, panel I). CD45 is a
human-specific pan-leukocyte marker. Human CD45+ cells
within gated region R2 were analyzed for multiple cell surface markers
to assess pluripotency of repopulating cells. Figure 5B (panel II)
demonstrates the presence of mature cells (CD15+) among
myeloid progeny (CD33+). As shown previously, B-lymphoid
cells are dominantly represented in the murine BM, as detected by
staining for CD19 and CD20 (Figure 5B, panel III).17 In
addition to differentiated human cells, a large proportion of
CD34+ cells was detected (Figure 5B, panel IV), which
provides evidence that immature cells are produced and maintained in
the murine BM in mice transplanted with this population. Similar
compositions of human hematopoietic engraftment were seen in animals
transplanted with CD7 cells within the
CD34+CD38Lin fraction
(data not shown). Taken together these results indicate that
CD34SRCs and CD34+SRCs share the same
AC133+CD7 phenotype. AC133 therefore may
provide a common cell surface marker to isolate both
CD34 and CD34+ human stem cells.
| |
Discussion |
The relationship of primitive cells within the human hematopoietic
hierarchy has been difficult to clarify due to the heterogeneity of the
stem cell compartment. This heterogeneity creates a major barrier in
the isolation of discrete populations among putative stem cells for
comparative analysis in functional assays. Using differences in the
cell surface expression of CD7 and AC133, we identified subpopulations
among both
CD34CD38Lin
and CD34+CD38Lin
cells that have distinct biological functions. Using in vitro and in
vivo assays, our results demonstrate that cell populations lacking CD7
but expressing AC133 (AC133+CD7) possessed
primitive hematopoietic activity unique to remaining CD3438Lin or
CD34+CD38Lin
populations. An extremely rare subset of
AC133+CD34CD38Lin
cells was identified at a frequency of 0.2% of the total
CD34CD38Lin
cells from human CB. This was the only subset among the
CD34CD38Lin
cells that possessed progenitor activity equivalent to that
of CD34+CD38Lin
cells, acquired cell surface expression of CD34, and was detected in
the BM of NOD/SCID mice 8 weeks after intravenous transplantation. Taken together, we suggest that this previously unidentified population contains primitive precursors of CD34+ cells and represents
the most highly purified fraction of primitive human
CD34 cells to date.
Until recently, characterization of primitive cell populations in the
human hematopoietic system was limited to those populations expressing
cell surface CD34.16 The isolation of highly purified cell
populations that do not express markers associated with lineage commitment, termed lineage negative (Lin), has
allowed for the comparison and investigation of populations that are
Lin but do not express CD34
(CD34Lin).16 Using in
vitro assays, human CD34Lin cells
have been postulated to have little to no progenitor capacity due to
their extreme immature nature and/or lack of appropriate growth factors
to support these novel cell populations.14,16 The ability
of cells within this large population to repopulate recipient animals,
however, demonstrated that this population was capable of hematopoietic
stem cell activity.16 We identified a rare population of
cells within the
CD34CD38Lin
population that expresses AC133 and possesses in vitro progenitor capacity equivalent to the most highly purified CD34+ human
stem cell fractions. The identification of these cells indicates that
primitive subsets of CD34Lin
cells are capable of being detected in vitro and respond to similar cytokines as CD34+Lin cells.
Furthermore, this same population is capable of producing CD34+ cells in serum-free liquid cultures.
Similar to previous studies using
CD34CD38Lin
cells, attempts to demonstrate repopulation capacity in NOD/SCID
mice from de novo isolated
AC133+CD34CD38Lin
or
AC133CD34CD38Lin
cells have been infrequent. In the case of the
AC133+CD34CD38Lin
subfraction, this may be attributed to the fact that the number of
cells available for transplantation is lower than the number of cells
required to successfully demonstrate repopulation using the NOD/SCID
assay or the number of cells required for ex vivo stimulation prior to
transplantation, as shown previously.16 Ex vivo culture of
the AC133+ subset from
CD34CD38Lin
cells allowed for a higher frequency of detection of human cells in
mice transplanted with the AC133+ population, which
indicates that CD34SRCs require prestimulation.
However, the levels of human chimerism in the BM of recipient mice from
CD34SRCs are low, even after ex vivo culture. Based
on the results of this study and previous work, it is clear that
detection of CD34SRCs in the NOD/SCID mouse is
inferior to the engraftment capacity of CD34+SRCs. An
appropriate facilitating population of cells, which differs from
accessory populations required for low numbers of CD34+
repopulating cells to engraft NOD/SCID mice, may be required for these
distinct CD34cells.26 The NOD/SCID assay
therefore requires further development to be useful for the future
characterization of human CD34 stem cells and to
fully elucidate the role of this cell in clinical stem cell transplants.
Previous studies have suggested that CD7 is a marker for lymphoid cells
and may also serve as a primitive stem cell marker because many acute
myeloid leukemic blasts coexpress CD7 and CD34.27,28 Our
results indicate that
CD34+CD38Lin cells
expressing CD7 had limited progenitor capacity within the myeloid
lineage, yet they failed to demonstrate repopulation capacity in
NOD/SCID mice. This was consistent with CD7
subfractions found in the
CD34CD38Lin
populations, and we propose that the bulk of these CD7+
cells within the
CD34CD38Lin
population are fully mature and fail to be removed during lineage depletion. AC133+ and AC133 cells
within the
CD34+CD38Lin
population demonstrated no detectable difference in the number or type
of progenitors. This is in contrast to previous studies in which
AC133+ demonstrated a higher frequency of total and
multipotent progenitors when compared with AC133
cells.29
CD34+CD38Lin cells
represent only 5%-8% of the total CD34+ cells. These
discrepancies may be due to the fact that both AC133+ and
AC133 cells were isolated from a more primitive and
homogenous population in this study and that they were derived
from human CB as opposed to adult BM. The
AC133+CD34+CD38Lin
population almost exclusively contains CD34+SRCs, which is
similar to previous studies demonstrating that the
AC133+CD34+ population from BM is enriched
for repopulating cells.29,30 All AC133+ cells
within the
CD34+CD38Lin fraction
are CD7, and our data indicates that human
CD34+SRCs are found exclusively in a more highly purified
population of
AC133+CD7CD34+CD38Lin
cells. This represents a significant refinement in the purification of
candidate human CD34+ stem cells.
Similar populations of AC133+CD34 cells
have been detected in human leukemias.31,32 Acute myeloid
leukemia (AML) patients present with both CD34+ and
AC133+CD34 blasts, and as a result,
these investigators31 hypothesized that the presence of
AC133+CD34 blasts may represent a
developmental transition in the hierarchy of hematopoiesis to
CD34+ blasts. AML has been shown to arise from a stem cell
population,33 and it is equally possible that some
leukemias occur from a transformation event in primitive
AC133+CD34 cells which leads to a clonal
expansion of leukemic stem cells. A proportion of these transformed
AC133+CD34 cells may have the capacity
to differentiate into CD34+ cells that lead to the
CD34+ leukemic clone. In this study, our
identification of a normal primitive population of
AC133+CD34 cells, which is capable of
producing CD34+ cells, supports this hypothesis. Therefore
it will be critical to isolate
AC133+CD34 cells from AML patients to
determine whether they have undergone transformation using
chromosomal/molecular diagnostic markers. Alternatively,
AC133+CD34 cells derived from AML
patients may have normal hematopoietic function and would then serve as
an ideal candidate for purging strategies during autologous transplantation.
Preliminary data from our laboratory indicates that
AC133+CD34Lin cells
are present in adult BM (data not shown). Thus, identification of a
population of cells within the
CD34CD38Lin
compartment that can be selected and quantitated using a positive marker may permit the diagnostic evaluation of human
CD34 stem cells in various clinical procedures.
These include both autologous and allogenic BM transplantation and the
ability to evaluate stem cell mobilization using this phenotype. In
addition, the
AC133+CD34CD38Lin
phenotype will assist in developing methods for gene transfer into this
population by first optimizing transduction efficiencies in the
clonogenic progeny of these cells using in vitro assays. AC133 is expressed on both repopulating CD34+ cells and
primitive CD34 subpopulations, and AC133 may,
therefore, provide a more appropriate method to enrich stem cells than
CD34 selection alone, thus preventing the discard of
CD34 subsets that could be critical in human
hematopoietic engraftment.
| |
Acknowledgments |
We thank Amgen Inc, Thousand Oaks, CA, for cytokines and the staff of
the labor and delivery departments of St Joseph's Hospital and London Health Sciences, London, Ontario, Canada, and
especially Marlene Watson and Jan Popma for providing cord blood
specimens. In addition, we would like to thank Dr C. Awaraji for his
technical support and Dr D. Kelvin for critically reviewing this manuscript.
| |
Footnotes |
Supported by a grant from the Cancer Research Society Inc,
Quebec, Canada; a grant (#MT-15063) from the Medical Research Council of Canada, Ontario, Canada; and a scholarship award (#MSH-35681) to
M.B. from the Medical Research Council of Canada, Ontario, Canada.
L. G. and B. M. contributed equally to this work.
Submitted June 28, 1999; accepted January 4, 2000.
Reprints: Mickie Bhatia, The John P. Robarts
Research Institute, 100 Perth Drive, London, Ontario, N6A 5K8 Canada; e-mail: mbhatia{at}rri.on.ca.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Morrison SJ, Uchida N, Weissman IL.
The biology of hematopoietic stem cells.
Annu Rev Cell Dev Biol.
1995;11:35-71[Medline]
[Order article via Infotrieve].
2.
Anderson WF.
Gene therapy.
Scientific American.
1995;273:124-128[Medline]
[Order article via Infotrieve].
3.
Moore MA.
Expansion of myeloid stem cells in culture.
Semin Hematol.
1995;32:183-200[Medline]
[Order article via Infotrieve].
4.
Orkin SH.
Hematopoiesis: how does it happen?
Curr Opin Cell Biol.
1995;7:870-877[Medline]
[Order article via Infotrieve].
5.
Petzer AL, Hogge DE, Landsdorp PM, Reid DS, Eaves CJ.
Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.
Proc Nat Acad Sci U S A.
1996;93:1470-1474[Abstract/Free Full Text].
6.
Petzer AL, Zandstra PW, Piret JM, Eaves CJ.
Differential cytokine effects on primitive (CD34+CD38) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin.
J Exp Med.
1996;183:2551-2558[Abstract/Free Full Text].
7.
Spangrude GJ, Smith L, Uchida N, et al.
Mouse hematopoietic stem cells.
Blood.
1991;78:1395-1402[Free Full Text].
8.
Baum CM, Weissman IL, Tsukamoto AS, Buckle A-S, Peault B.
Isolation of a candidate human hematopoietic stem-cell population.
Proc Natl Acad Sci U S A.
1992;89:2804-2808[Abstract/Free Full Text].
9.
Morrison SJ, Wandycz AM, Hemmati HD, Wright DE, Weissman IL.
Identification of a lineage of multipotent hematopoietic progenitors.
Development.
1997;124:1929-1939[Abstract].
10.
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC.
Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.
J Exp Med.
1996;183:1797-1806[Abstract/Free Full Text].
11.
Jones RJ, Collector MI, Barber JP, et al.
Characterization of mouse lymphohematopoietic stem cells lacking spleen colony-forming activity.
Blood.
1996;88:487-491[Abstract/Free Full Text].
12.
Osawa M, Hanada K, Hamada H, Nakauchi H.
Long-term lymphohematopoietic reconstitution by a single CD34 investigative hematopoietic stem cell.
Science.
1996;273:242-245[Abstract].
13.
Spangrude GJ.
Enrichment of murine haemopoietic stem cells: Diverging roads.
Immunol Today.
1989;10:344-350[Medline]
[Order article via Infotrieve].
14.
Goodell MA, Rosenzweig M, Kim H, et al.
Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.
Nat Med.
1997;3:1337-1345[Medline]
[Order article via Infotrieve].
15.
Zanjani ED, Almeida-Porada G, Livingston AG, Flake AW, Ogawa M.
Human bone marrow CD34 cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells [see comments].
Exp Hematol.
1998;26:353-360[Medline]
[Order article via Infotrieve].
16.
Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE.
A newly discovered class of human hematopoietic cells with SCID-repopulating activity.
Nat Med.
1998;4:1038-1045[Medline]
[Order article via Infotrieve].
17.
Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE.
Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice.
Proc Natl Acad Sci U S A.
1997;94:5320-5325[Abstract/Free Full Text].
18.
Miraglia S, Godfrey W, Yin AH, et al.
A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning.
Blood.
1997;90:5013-5021[Abstract/Free Full Text].
19.
Miraglia S, Godfrey W, Buck D.
A response to AC133 hematopoietic stem cell antigen: human homologue of mouse kidney prominin or distinct member of a novel protein family? [letter].
Blood.
1998;91:4390-4391[Free Full Text].
20.
Corbeil D, Roper K, Weigmann A, Huttner WB.
AC133 hematopoietic stem cell antigen: human homologue of mouse kidney prominin or distinct member of a novel protein family? [letter].
Blood.
1998;91:2625-2626[Free Full Text].
21.
Bhatia M, Bonnet D, Kapp U, Wang JC, Murdoch B, Dick JE.
Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.
J Exp Med.
1997;186:619-624[Abstract/Free Full Text].
22.
Hogan CJ, Shpall EJ, McNulty O, et al.
Engraftment and development of human CD34(+)-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice.
Blood.
1997;90:85-96[Abstract/Free Full Text].
23.
Larochelle A, Vormoor J, Hanenberg H, et al.
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.
Nat Med.
1996;2:1329-1337[Medline]
[Order article via Infotrieve].
24.
Huang S, Terstappen LW.
Lymphoid and myeloid differentiation of single human CD34+, HLA-DR+, CD38 hematopoietic stem cells.
Blood.
1994;83:1515-1526[Abstract/Free Full Text].
25.
Craig W, Kay R, Cutler RB, Lansdorp PM.
Expression of Thy-1 on human hematopoietic progenitor cells.
J Exp Med.
1993;177:1331-1342[Abstract/Free Full Text].
26.
Bonnet D, Bhatia M, Wang JC, Kapp U, Dick JE.
Cytokine treatment or accessory cells are required to initiate engraftment of purified primitive human hematopoietic cells transplanted at limiting doses into NOD/SCID mice.
Bone Marrow Transplant.
1999;23:203-209[Medline]
[Order article via Infotrieve].
27.
Tokunaga Y, Miyamoto T, Okamura T, et al.
Effect of thrombopoietin on proliferation of blasts from CD7-positive acute myelogenous leukaemia.
Br J Haematol.
1998;102:1232-1240[Medline]
[Order article via Infotrieve].
28.
Saxena A, Sheridan DP, Card RT, McPeek AM, Mewdell CC, Skinnider LF.
Biologic and clinical significance of CD7 expression in acute myeloid leukemia.
Am J Hematol.
1998;58:278-284[Medline]
[Order article via Infotrieve].
29.
Yin AH, Miraglia S, Zanjani ED, et al.
AC133, a novel marker for human hematopoietic stem and progenitor cells.
Blood.
1997;90:5002-5012[Abstract/Free Full Text].
30.
de Wynter EA, Buck D, Hart C, et al.
CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors.
Stem Cells.
1998;16:387-396[Abstract/Free Full Text].
31.
Horn PA, Tesch H, Staib P, Kube D, Diehl V, Voliotis D.
Expression of AC133, a novel hematopoietic precursor antigen, on acute myeloid leukemia cells [letter].
Blood.
1999;93:1435-1437[Free Full Text].
32.
Kratz-Albers K, Zuhlsdorp M, Leo R, Berdel WL, Buchner T, Serve H.
Expression of a AC133, a novel stem cell marker, on human leukemic blasts lacking CD34 antigen and on a human CD34+ leukemic line: MUTZ-2 [letter].
Blood.
1998;92:4485-4487[Free Full Text].
33.
Bonnet D, Dick JE.
Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.
Nat Med.
1997;3:730-737[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. V. Cox, P. Diamanti, R. S. Evely, P. R. Kearns, and A. Blair
Expression of CD133 on leukemia-initiating cells in childhood ALL
Blood,
April 2, 2009;
113(14):
3287 - 3296.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Shi, M. Ishikawa, N. Kamei, T. Nakasa, N. Adachi, M. Deie, T. Asahara, and M. Ochi
Acceleration of Skeletal Muscle Regeneration in a Rat Skeletal Muscle Injury Model by Local Injection of Human Peripheral Blood-Derived CD133-Positive Cells
Stem Cells,
April 1, 2009;
27(4):
949 - 960.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. P. Kusumbe, A. M. Mali, and S. A. Bapat
CD133-Expressing Stem Cells Associated with Ovarian Metastases Establish an Endothelial Hierarchy and Contribute to Tumor Vasculature
Stem Cells,
March 1, 2009;
27(3):
498 - 508.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-D. Minana, F. Carbonell-Uberos, V. Mirabet, S. Marin, and A. Encabo
IFATS Collection: Identification of Hemangioblasts in the Adult Human Adipose Tissue
Stem Cells,
October 1, 2008;
26(10):
2696 - 2704.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. van Pel, H. Hagoort, J. Hamann, and W. E. Fibbe
CD97 is differentially expressed on murine hematopoietic stem-and progenitor-cells
Haematologica,
August 1, 2008;
93(8):
1137 - 1144.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Langer, M. D. Radmacher, A. S. Ruppert, S. P. Whitman, P. Paschka, K. Mrozek, C. D. Baldus, T. Vukosavljevic, C.-G. Liu, M. E. Ross, et al.
High BAALC expression associates with other molecular prognostic markers, poor outcome, and a distinct gene-expression signature in cytogenetically normal patients younger than 60 years with acute myeloid leukemia: a Cancer and Leukemia Group B (CALGB) study
Blood,
June 1, 2008;
111(11):
5371 - 5379.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Hess, T. P. Craft, L. Wirthlin, S. Hohm, P. Zhou, W. C. Eades, M. H. Creer, M. S. Sands, and J. A. Nolta
Widespread Nonhematopoietic Tissue Distribution by Transplanted Human Progenitor Cells with High Aldehyde Dehydrogenase Activity
Stem Cells,
March 1, 2008;
26(3):
611 - 620.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. K. Burt, Y. Loh, W. Pearce, N. Beohar, W. G. Barr, R. Craig, Y. Wen, J. A. Rapp, and J. Kessler
Clinical Applications of Blood-Derived and Marrow-Derived Stem Cells for Nonmalignant Diseases
JAMA,
February 27, 2008;
299(8):
925 - 936.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. C. Schatteman, M. Dunnwald, and C. Jiao
Biology of bone marrow-derived endothelial cell precursors
Am J Physiol Heart Circ Physiol,
January 1, 2007;
292(1):
H1 - H18.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G Perrella, P Brusini, R Spelat, P Hossain, A Hopkinson, and H S Dua
Expression of haematopoietic stem cell markers, CD133 and CD34 on human corneal keratocytes
Br. J. Ophthalmol.,
January 1, 2007;
91(1):
94 - 99.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Suzuki, Y. Yokoyama, K. Kumano, M. Takanashi, S. Kozuma, T. Takato, T. Nakahata, M. Nishikawa, S. Sakano, M. Kurokawa, et al.
Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1-Fc Chimeric Protein
Stem Cells,
November 1, 2006;
24(11):
2456 - 2465.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Goussetis, A. Manginas, M. Koutelou, I. Peristeri, M. Theodosaki, N. Kollaros, E. Leontiadis, A. Theodorakos, G. Paterakis, G. Karatasakis, et al.
Intracoronary Infusion of CD133+ and CD133-CD34+ Selected Autologous Bone Marrow Progenitor Cells in Patients with Chronic Ischemic Cardiomyopathy: Cell Isolation, Adherence to the Infarcted Area, and Body Distribution
Stem Cells,
October 1, 2006;
24(10):
2279 - 2283.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Goussetis, M. Theodosaki, G. Paterakis, C. Tsecoura, and S. Graphakos
In Vitro Identification of a Cord Blood CD133+CD34-Lin+ Cell Subset that Gives Rise to Myeloid Dendritic Precursors
Stem Cells,
April 1, 2006;
24(4):
1137 - 1140.
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. J. Suuronen, S. Wong, V. Kapila, G. Waghray, S. C. Whitman, T. G. Mesana, and M. Ruel
Generation of CD133+ cells from CD133- peripheral blood mononuclear cells and their properties
Cardiovasc Res,
April 1, 2006;
70(1):
126 - 135.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Jaatinen, H. Hemmoranta, S. Hautaniemi, J. Niemi, D. Nicorici, J. Laine, O. Yli-Harja, and J. Partanen
Global Gene Expression Profile of Human Cord Blood-Derived CD133+ Cells
Stem Cells,
March 1, 2006;
24(3):
631 - 641.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Hess, L. Wirthlin, T. P. Craft, P. E. Herrbrich, S. A. Hohm, R. Lahey, W. C. Eades, M. H. Creer, and J. A. Nolta
Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells
Blood,
March 1, 2006;
107(5):
2162 - 2169.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Toren, B. Bielorai, J. Jacob-Hirsch, T. Fisher, D. Kreiser, O. Moran, S. Zeligson, D. Givol, A. Yitzhaky, J. Itskovitz-Eldor, et al.
CD133-Positive Hematopoietic Stem Cell "Stemness" Genes Contain Many Genes Mutated or Abnormally Expressed in Leukemia
Stem Cells,
September 1, 2005;
23(8):
1142 - 1153.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. J. Pearce, D. Taussig, C. Simpson, K. Allen, A. Z. Rohatiner, T. A. Lister, and D. Bonnet
Characterization of Cells with a High Aldehyde Dehydrogenase Activity from Cord Blood and Acute Myeloid Leukemia Samples
Stem Cells,
June 1, 2005;
23(6):
752 - 760.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Fang, C. Zheng, L. Liao, Q. Han, Z. Sun, X. Jiang, and R. C. H. Zhao
Identification of human chronic myelogenous leukemia progenitor cells with hemangioblastic characteristics
Blood,
April 1, 2005;
105(7):
2733 - 2740.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Armstrong, M. Stojkovic, I. Dimmick, S. Ahmad, P. Stojkovic, N. Hole, and M. Lako
Phenotypic Characterization of Murine Primitive Hematopoietic Progenitor Cells Isolated on Basis of Aldehyde Dehydrogenase Activity
Stem Cells,
December 1, 2004;
22(7):
1142 - 1151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Hess, T. E. Meyerrose, L. Wirthlin, T. P. Craft, P. E. Herrbrich, M. H. Creer, and J. A. Nolta
Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity
Blood,
September 15, 2004;
104(6):
1648 - 1655.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. J. Summers, C. M. Heyworth, E. A. de Wynter, C. A. Hart, J. Chang, and N. G. Testa
AC133+ G0 Cells from Cord Blood Show a High Incidence of Long-Term Culture-Initiating Cells and a Capacity for More Than 100 Million-Fold Amplification of Colony-Forming Cells In Vitro
Stem Cells,
September 1, 2004;
22(5):
704 - 715.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. C. Dooley, B. K. Oppenlander, and M. Xiao
Analysis of Primitive CD34- and CD34+ Hematopoietic Cells from Adults: Gain and Loss of CD34 Antigen by Undifferentiated Cells Are Closely Linked to Proliferative Status in Culture
Stem Cells,
July 1, 2004;
22(4):
556 - 569.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Denning-Kendall, S. Singha, B. Bradley, and J. Hows
Cobblestone Area-Forming Cells in Human Cord Blood Are Heterogeneous and Differ from Long-Term Culture-Initiating Cells
Stem Cells,
November 1, 2003;
21(6):
694 - 701.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Rutella, G. Bonanno, M. Marone, D. de Ritis, A. Mariotti, M. T. Voso, G. Scambia, S. Mancuso, G. Leone, and L. Pierelli
Identification of a Novel Subpopulation of Human Cord Blood CD34-CD133-CD7-CD45+Lineage- Cells Capable of Lymphoid/NK Cell Differentiation After In Vitro Exposure to IL-15
J. Immunol.,
September 15, 2003;
171(6):
2977 - 2988.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Murdoch, K. Chadwick, M. Martin, F. Shojaei, K. V. Shah, L. Gallacher, R. T. Moon, and M. Bhatia
Wnt-5A augments repopulating capacity and primitive hematopoietic development of human blood stem cells invivo
PNAS,
March 18, 2003;
100(6):
3422 - 3427.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Yasui, K. Matsumoto, F. Hirayama, Y. Tani, and T. Nakano
Differences Between Peripheral Blood and Cord Blood in the Kinetics of Lineage-Restricted Hematopoietic Cells: Implications for Delayed Platelet Recovery Following Cord Blood Transplantation
Stem Cells,
March 1, 2003;
21(2):
143 - 151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Kuci, J. T. Wessels, H.-J. Buhring, K. Schilbach, M. Schumm, G. Seitz, J. Loffler, P. Bader, P. G. Schlegel, D. Niethammer, et al.
Identification of a novel class of human adherent CD34- stem cells that give rise to SCID-repopulating cells
Blood,
February 1, 2003;
101(3):
869 - 876.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Guo, M. Lubbert, and M. Engelhardt
CD34- Hematopoietic Stem Cells: Current Concepts and Controversies
Stem Cells,
January 1, 2003;
21(1):
15 - 20.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. E. Jay, L. Gallacher, and M. Bhatia
Emergence of muscle and neural hematopoiesis in humans
Blood,
October 16, 2002;
100(9):
3193 - 3202.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Hess, K. D. Levac, F. N. Karanu, M. Rosu-Myles, M. J. White, L. Gallacher, B. Murdoch, M. Keeney, P. Ottowski, R. Foley, et al.
Functional analysis of human hematopoietic repopulating cells mobilized with granulocyte colony-stimulating factor alone versus granulocyte colony-stimulating factor in combination with stem cell factor
Blood,
July 18, 2002;
100(3):
869 - 878.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Testa, G. F. Torelli, R. Riccioni, A. O. Muta, S. Militi, L. Annino, G. Mariani, A. Guarini, S. Chiaretti, J. Ritz, et al.
Human acute stem cell leukemia with multilineage differentiation potential via cascade activation of growth factor receptors
Blood,
May 29, 2002;
99(12):
4634 - 4637.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Feuring-Buske and D. E. Hogge
Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34+CD38{-} progenitor cells from patients with acute myeloid leukemia
Blood,
June 15, 2001;
97(12):
3882 - 3889.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. N. Karanu, B. Murdoch, T. Miyabayashi, M. Ohno, M. Koremoto, L. Gallacher, D. Wu, A. Itoh, S. Sakano, and M. Bhatia
Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells
Blood,
April 1, 2001;
97(7):
1960 - 1967.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Audet, C. L. Miller, S. Rose-John, J. M. Piret, and C. J. Eaves
Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells
PNAS,
February 13, 2001;
98(4):
1757 - 1762.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. N. Karanu, B. Murdoch, L. Gallacher, D. M. Wu, M. Koremoto, S. Sakano, and M. Bhatia
The Notch Ligand Jagged-1 Represents a Novel Growth Factor of Human Hematopoietic Stem Cells
J. Exp. Med.,
November 6, 2000;
192(9):
1365 - 1372.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. H. S. Kabarowski and O. N. Witte
Consequences of BCR-ABL Expression within the Hematopoietic Stem Cell in Chronic Myeloid Leukemia
Stem Cells,
November 1, 2000;
18(6):
399 - 408.
[Abstract]
[Full Text]
|
|
|
|
|
Top
Abstract
Introduction