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Blood, 15 September 2000, Vol. 96, No. 6, pp. 2125-2133
HEMATOPOIESIS
Hoechst dye efflux reveals a novel
CD7+CD34 lymphoid
progenitor in human umbilical cord blood
Robert W. Storms,
Margaret A. Goodell,
Alan Fisher,
Richard C. Mulligan, and
Clay Smith
From the Center for Genetic and Cellular Therapies,
Division of Experimental Surgery, Department of Surgery, Duke
University Medical Center; the Duke Comprehensive Cancer Center, Duke
University Medical Center; and the Division of Oncology and
Transplantation; Duke University Medical Center, Durham, NC; and the
Gene Therapy Program, Children's Hospital, Harvard Medical School,
Boston, MA.
 |
Abstract |
A novel Hoechst 33342 dye efflux assay was recently developed that
identifies a population of hematopoietic cells termed side population
(SP) cells. In the bone marrow of multiple species, including
mice and primates, the SP is composed primarily of CD34
cells, yet has many of the functional properties of hematopoietic stem cells (HSCs). This report characterizes SP cells from human umbilical cord blood (UCB). The SP in unfractionated UCB was enriched for CD34+ cells but also contained a large population of
CD34 cells, many of which were mature lymphocytes. SP
cells isolated from UCB that had been depleted of lineage-committed
cells (Lin UCB) contained CD34+ and
CD34 cells in approximately equivalent proportions.
Similar to previous descriptions of human HSCs, the
CD34+Lin SP cells were
CD38dimHLA-DRdimThy-1dimCD45RACD71
and were enriched for myelo-erythroid precursors. In contrast, the
CD34Lin SP cells were
CD38HLA-DRThy-1CD71
and failed to generate myelo-erythroid progeny in vitro. The majority
of these cells were
CD7+CD11b+CD45RA+, as might be
expected of early lymphoid cells, but did not express other lymphoid
markers. The CD7+CD34Lin UCB SP
cells did not proliferate in simple suspension cultures but did
differentiate into natural killer cells when cultured on stroma with
various cytokines. In conclusion, the human Lin UCB SP
contains both CD34+ multipotential stem cells and a
novel CD7+CD34Lin lymphoid
progenitor. This observation adds to the growing body of evidence
that CD34 progenitors exist in humans.
(Blood. 2000;96:2125-2133)
© 2000 by The American Society of Hematology.
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Introduction |
The characterization and manipulation of
hematopoietic stem cells (HSCs) for transplantation and gene therapy
purposes has been intensely studied in recent years.1,2
The most primitive HSCs have an extensive potential for self-renewal
and can give rise to all blood-cell lineages.3
A number of in vitro and xenochimeric transplantation studies have
demonstrated that primitive human HSCs express CD34, a cell-surface
protein of unknown function.4-6 A small number of primate
studies have confirmed that CD34+ bone marrow cells can
durably reconstitute multilineage hematopoiesis following
transplantation.7 On the basis of this evidence, a number
of autologous and allogeneic clinical bone marrow transplantation trials have used CD34+-enriched cell
preparations.8 In these trials, multilineage hematopoietic engraftment occurred in the majority of patients, further
supporting the contention that HSCs express CD34.
Recently, several experimental observations have suggested that HSCs
that do not express CD34 may also exist. Murine CD34lo/
HSCs with long-term repopulating ability have been isolated from LinSca-1+ fractions of bone
marrow.9,10 Similarly, murine CD34lo/ HSCs
have been fractionated from bone marrow by virtue of their aldehyde
dehydrogenase activity or by virtue of their capacity to efflux various
dyes.11,12 One recent study has indicated that the
majority of HSCs in resting murine bone marrow are
CD34lo/ but that CD34 expression on HSCs is increased
following cell activation.13 This observation raises the
possibility that expression of CD34 on HSCs is dynamic and may vary
depending on the physiologic status of the donor.14
In humans, the expression pattern of CD34 on HSCs is less clear because
of the absence of simple and authentic human transplant models. Human
CD34 cells have reconstituted multiple hematopoietic cell
lineages in xenotransplant animal models including fetal sheep and
nonobese diabetic/severe combined immune-deficient (NOD/SCID)
mice.15,16 In addition, a subset of
LinCD34 cells isolated from both bone
marrow and mobilized peripheral blood gave rise to long-term culture
(LTC)-initiating cells following incubation with high doses
of certain cytokines.17 Finally, a
LinCD34 population has been shown to
acquire CD34 expression, clonogenic activity, and NOD/SCID repopulating
activity following a brief period in culture on stroma supplemented
with several cytokines.18 Although these studies provide
provocative evidence that CD34 HSCs may exist in humans,
these cells have been difficult to study because they are obscured by a
much larger number of mature CD34 cells. In addition, the
Lin CD34 populations studied to date appear
to be heterogeneous so that multiple-lineage-committed progenitors
rather than individual multilineage HSCs may have generated the
observed stem cell activity.
A recently developed technique for isolating HSCs based on Hoechst
33342 dye efflux may prove useful for addressing these issues.12,19 This technique identifies a small
subpopulation of cells, termed the side population (SP), on the basis
of its unique fluorescence emission properties. In mice, bone marrow SP
cells had the phenotypic and functional properties of HSCs. These cells
were highly enriched for long-term hematopoietic reconstitution activity despite expressing low to no levels of CD34.12,19 SP cells have also been identified in primate and human bone marrow and
in human umbilical cord blood (UCB).19 CD34
SP cells isolated from rhesus bone marrow lacked the expression of
obvious lineage commitment markers, acquired CD34 expression in vitro,
were enriched for cells capable of generating clonogenic progeny in
LTCs, and could generate T-cells on rhesus thymic
stroma.19
These observations suggest that using Hoechst dye efflux to isolate SP
cells may be a useful method for enriching rare populations of
CD34 HSCs. Since UCB is increasingly being used for
transplantation purposes,20,21 we sought to characterize
UCB SP cells and to determine whether the UCB SP contains primitive
CD34 hematopoietic cells.
| |
Materials and methods |
UCB processing
Staff members of the Carolina Cord Blood Bank collected human
UCB after obtaining institutional-review-board-approved informed consent. UCB was diluted with Dulbecco's phosphate-buffered saline (PBS), and red blood cells were agglutinated at room temperature with
the use of Hespan (DuPont Pharma, Wilmington, DE) at a final concentration of 1%. Nonagglutinated white blood cells were harvested, and residual red cells were hemolyzed at 37°C in 0.17 mol/L
NH4Cl, 20 mmol/L Tris-HCl, pH 7.2, and 200 µmol/L EDTA.
Lineage-committed cells were then removed from the white-cell fraction
with the use of the CD34+ StemSep enrichment cocktail
(StemCell Technologies Inc, Vancouver, British Columbia, Canada)
according to kit instructions. The recovered cells, termed
Lin cells, were washed in Iscove's Modified Dulbecco's
Medium (IMDM)/10% fetal calf serum (FCS) and then held on ice until
they were analyzed or further fractionated with fluorescence-activated
cell sorting (FACS).
Hoechst 33342 and antibody staining
To identify and isolate SP cells, undepleted or
Lin UCB was resuspended at 106 cells per
milliliter in IMDM/2% FCS (staining media) and preincubated at 37°C
for 30 minutes. The cells were then labeled with 2.5 µg/mL Hoechst 33342 (Molecular Probes; Eugene, OR) in staining media at
37°C for 90 minutes. When verapamil (American Regent Laboratories; Shirley, NY) was used, it was included at 50 µM. After
staining, the cells were washed, resuspended in ice-cold staining
media, and then maintained on ice until their analysis or sorting. For staining of cell-surface antigens, cells were resuspended in 100 µL of staining media, and antibodies were added directly to
the cell suspensions. The cells were incubated on ice for 15 minutes and then washed with ice-cold staining media. Immediately prior to FACS
analysis or sorting, 1 to 2 µg/mL propidium iodide (PI) (Sigma
Chemical, St Louis, MO) was added to the cell suspensions.
Directly conjugated fluorescent antibodies used in these studies were
antibodies directed against CD2 (clone S5.2), CD3 (clone SK7), CD5
(clone L17F12), CD7 (clone 4H9), CD16 (clone NKP15), CD34 (clone 8G12,
ie, HPCA-2), CD38 (clone HB7), and CD56 (clone MY31) from Becton
Dickinson Immunocytometry Systems (BDIS) (San Jose, CA). In addition,
an anti-CD7 (CD7-6B7) antibody was obtained from CalTag Laboratories
(Burlingame, CA); anti-CD45 (clones KC-56 and J33) as well as pooled
anti-CD34 antibodies (clones QBEnd10, Immu-133, and Immu-134) were
obtained from Coulter/Immunotech (Miami, FL); an anti-CD94 antibody
(clone HP-3D9) was obtained from PharMingen (San Diego, CA); an
anti-CD38 antibody (clone B-A6) was obtained from BioSource
International (Camarillo, CA); and an anti-CD45RA antibody (clone
F8-11-13) was obtained from Southern Biotechnology Associates, Inc
(Birmingham, AL).
Fluorescence-activated cell sorting
Cells were analyzed and sorted on a FACStar Plus (BDIS) cell
sorter equipped with dual Coherent I-90 lasers. Hoechst 33342 was
excited at 351 nm, and fluorescence emission was detected with the use
of 450DF20 (blue) and LP675 (far-red) filters (Omega Optical Inc,
Brattleboro, VT). A 610-nm short pass dichroic mirror was used to
separate these emission wavelengths (Omega Optical Inc). Fluorescence
from the Hoechst dye was acquired in linear scales. Dead and dying
cells were excluded on the basis of PI uptake. Fluorochrome-conjugated
antibodies were excited at 488 nm, and their fluorescence emission was
detected by means of standard filters.
In some experiments, CD34+ and CD34 SP cells
were isolated directly from unfractionated UCB that had been stained
simultaneously with Hoechst and phycoerythrin (PE)-conjugated
anti-CD34. For these assays, the SP was defined as 0.02% to 0.03% of
the total white-cell content of the unfractionated UCB. To isolate
CD34+ and CD34 fractions from the
Lin SP, multiple sequential sorts were employed to
optimize purity. After lineage depletion, the cells were stained with
Hoechst, and the SP region was defined on the cytometer on the basis of its low fluorescence emission in both blue and far-red wavelengths (representative profiles appear in Figure 3). SP cells were sorted as
the dimmest 1% of the Hoechst-stained Lin UCB. For
assays for myeloid growth, the Lin SP cells were then
washed and restained with anti-CD34 PE and anti-CD38 fluorescein
isothiocyanate (FITC) and re-sorted to isolate CD34+CD38dimLin SP and
CD34CD38dimLin SP fractions.
The CD38 gate was defined on the basis of a signal
intensity equivalent to, or less than, an FITC isotype control. On
reanalysis, purified CD34Lin SP cells did
not express CD34 as defined by monoclonal antibodies raised against
multiple different epitopes of the protein. For in vitro lymphoid
assays, the Lin SP cells were stained with anti-CD7 FITC,
with anti-CD34 allophycocyanin, and simultaneously with
anti-CD2 PE, CD3 PE, CD5 PE, and CD56 PE. The cells were then re-sorted
to isolate CD7+CD34Lin SP and
CD34+CD7±Lin SP subfractions.
Short-term and long-term colony-forming unit assays
Hematopoietic progenitor colony assays (HPCAs) were performed by
plating 100 to 200 cells in MethoCult H4431 containing
agar-leukocyte-conditioned media and recombinant human erythropoietin
(StemCell Technologies). The cells were incubated in a humidified
chamber at 37°C with 5% CO2, and hematopoietic colonies
(greater than 100 cells) were scored at 14 to 18 days after the
cultures were initiated. LTC assays were maintained on either
irradiated allogeneic bone marrow stroma or MS5 cells (graciously
provided by Dr Tadashi Sudo of the Kirin Pharmaceutical Research
Laboratory, Gunma, Japan). The MS5 stromal layers were established by
seeding 24-well plates (Corning Costar Corp, Cambridge, MA) with 6 to
7 × 104 MS5 cells per well in DMEM/10% FCS. Allogeneic
bone marrow stromal cells were seeded at similar densities in Myelocult
H5100 (StemCell Technologies) containing 1 µmol/L hydrocortisone
(succinate salt; Sigma Chemical). Stromal cells were cultured at 37°C
in a humidified incubator until the cultures approached approximately
80% confluence. The monolayers were then  -irradiated from a cesium
source (30 to 40 Gy for MS5 stromal layers; 17.5 Gy for allogeneic bone
marrow stroma). LTCs established with SP cells derived from
unfractionated UCB were initiated with 150 to 350 cells per well. LTCs
established from SP fractions derived from Lin UCB were
initiated with 400 to 2000 hematopoietic progenitor cells per well on
the irradiated MS5 cells. LTCs on MS5 cells were maintained in
Myelocult H5100 medium (StemCell Technologies) at 33°C in a
humidified chamber with 5% CO2. LTCs on allogeneic stroma
were maintained in Myelocult H5100 medium supplemented with 1 µmol/L
hydrocortisone, 25 ng/mL Kit ligand (KL), 10 ng/mL interleukin 3 (IL-3), and 10 ng/mL IL-6 (R&D Systems, Minneapolis, MN) at
37°C in a humidified chamber with 5% CO2. For all LTCs, half the media from each well were removed at weekly intervals and
replenished with fresh media. Adherent and nonadherent cells were
harvested after 5 weeks and plated into HPCAs as described above.
Lymphoid cell cultures
For lymphocyte suspension cultures, 100 to 500 sorted cells were
plated in duplicate either in serum-free media (BIT 9500, StemCell
Technologies) or in RPMI 1640/10% FCS with IL-2 (100 U/mL), IL-7 (10 ng/mL, R&D Systems), or IL-12 (10 ng/mL; R&D Systems). In some
cultures, 10% conditioned medium from phytohemagglutinin [PHA]-stimulated leukocytes (T-Stim without PHA;
Collaborative Biomedical Products, Bedford, MA) was added. For lymphoid
development on stroma, Lin SP cells were seeded onto
-irradiated (20 Gy) MS5 stroma at 100 to 2000 cells per well. Cells
were cultured either in MEM medium supplemented with 10% FCS or in
HAMS F12 medium supplemented with 1% bovine serum albumin,
2% FCS, 1 µmol/L ZnSO4, 1 µmol/L CuSO4, 5 µmol/L  -mercaptoethanol, and a mixture of insulin, transferrin, and selenium (ITS-G; Gibco BRL, Gaithersburg, MD). These
cultures were supplemented with KL, Flt3 ligand (F3L), IL-2,
IL-7, and/or IL-15 (all from R&D Systems), as described in Table
1.
Flow cytometric assay for natural killer cell function
A protocol for natural killer (NK) cell function, which measures
target cell death through the uptake of membrane impermeable DNA dyes,
was modified to evaluate NK-cell function in small numbers of
cells.22 The target cells used in these assays included
NK-sensitive K562 cells as well as NK-resistant Raji cells. K562 cells
were labeled with 3 µmol/L carboxyfluorescein succinimidylester
(CFSE) (Molecular Probes) in PBS at 106 cells per
milliliter for 10 minutes at room temperature, and Raji cells were
labeled with 0.5 µmol/L CFSE in a similar fashion. These
concentrations of CFSE achieved similar fluorescence intensities for
the 2 target cell lines. After CFSE labeling, the target cells were
incubated overnight at 37°C with 5% CO2 in RPMI 1640 medium supplemented with 10% FCS. For lysis assays, the target cells were plated in 50 µL RPMI 1640/10% FCS to deliver 1000 cells per well in 96-well V-bottom culture plates. Effector cells were isolated from the lymphoid development cultures (see above) by FACS sorting human CD45+ cells from the murine MS5 stroma. The sorted
cells were collected into RPMI 1640/10% FCS supplemented with 1000 U/mL IL-2 and plated in triplicate at the various effector-to-target
ratios described in the Figure legends. To facilitate cell-to-cell
interactions, the microtiter plates were centrifuged briefly at
700g. The cocultures were then incubated at 37°C with 5%
CO2. After 4 hours in coculture, 0.5 µg 7-AAD (Molecular
Probes) was delivered to each well in 50 µL RPMI 1640/10% FCS. After
an additional 45 to 60 minutes, the cells were pelleted, washed once
with PBS/2% FCS, and fixed in 1% formaldehyde prepared in
PBS/2% FCS.
| |
Results |
A Hoechst 33342 SP is present in human umbilical
cord blood
To identify SP cells in human UCB, initial studies were conducted
to establish an optimal Hoechst dye concentration and staining duration
(data not shown). Several conditions produced a similar pattern;
however, incubation of UCB with 2.5 µg/mL of Hoechst for 90 minutes
consistently identified a population of cells with a staining and
fluorescence-emission pattern similar to that of murine bone marrow SP
(Figure 1A, and data not shown). Goodell et al12 had previously shown that the Hoechst SP profile
in murine bone marrow was blocked by staining in the presence of verapamil, indicating that the dim staining of SP cells was at least
partially due to the efflux of Hoechst by a multidrug resistance (MDR)-like protein. The Hoechst staining of human UCB was also sensitive to verapamil (Figure 1B). The verapamil-sensitive UCB SP
subpopulation represented 0.40% ± 0.29% (n = 28) of the total white-cell content of UCB. Because in murine studies, the dimmest SP
cells had the highest capacity for long-term hematopoietic reconstitution, in subsequent UCB studies the dimmest 0.05% to 0.1%
of the total mononuclear-cell content was defined as the human
UCB SP (Figure 1A-B). In all cases, the fluorescent staining of the SP
cells was easily distinguishable from the majority of the UCB
cells.
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| Figure 1.
Hoechst dye efflux by human UCB.
The SP cells, as defined by verapamil-sensitive cells, are
indicated in the enclosed box. The data are representative of 28 experiments. (A) The Hoechst 33342 staining and emission patterns of
human UCB in the absence of verapamil. (B) The Hoechst 33342 staining
and emission patterns of human UCB in the presence of verapamil.
|
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To characterize the cells within the SP, unfractionated UCB was stained
with Hoechst in conjunction with antibodies directed against a variety
of cell-surface markers. Cell surface antigens for mature myeloid
cells, B cells, and erythroid cells were absent on UCB SP cells (Table
2). In
contrast, relatively high proportions of the CD34 UCB SP
cells expressed markers commonly found on mature lymphoid cells,
including CD2, CD3, CD5, CD16, and CD56 (Table 2). These antigens were
detected exclusively on the CD34 SP cells. The UCB SP
contained 11.7% ± 8.7% CD34+ cells and was
approximately 6-fold enriched for these cells relative to
unfractionated UCB (Figure 2A-B; Table
3). Similarly, the UCB SP contained
3.05% ± 2.9% CD34+CD38lo/ cells and was
approximately 15-fold enriched for these cells over the unfractionated
UCB (Figure 2A-B; Table 3). Despite this, 88.3% ± 8.7% of the UCB
SP cells were CD34. To determine whether the
CD34+ or the CD34 fractions of the UCB SP
contained any progenitor-cell activity, CD34+ SP and
CD34 SP cells were isolated and placed into LTC
colony-forming unit assays. Whereas the CD34+ SP fraction
was highly enriched in LTC progenitors relative to unfractionated UCB,
the CD34 SP contained only a very low proportion of LTC
progenitors (Figure 2C).
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| Figure 2.
Characterization of the UCB SP for properties of
HSCs.
(A,B) The expression of CD34 and CD38 on unfractionated UCB (A) and the
UCB SP (B). The SP represented the dimmest 0.05% to 0.1% of the
Hoechst-stained UCB. Quadrant statistics are provided for the
individual experiment depicted. These data are representative of 5 experiments. (C) Hematopoietic progenitors from unfractionated UCB,
CD34+ UCB, CD34+ UCB SP cells, and
CD34 UCB SP cells were enumerated in LTCs on allogeneic
bone marrow stroma. The data represent the mean ± SD of 3 experiments. Cultures of CD34+ UCB SP cells represent only
2 UCB samples; LTCs detected from CD34 SP cells
were derived from 1 well of 8 wells plated.
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The Lin SP contains
CD34+ and CD34
cells
One potential explanation for the low frequency of detectable
progenitors within the CD34 SP is that they may have been
diluted by the mature cells within the SP. To enrich for possible
primitive CD34 cells, the lineage-committed cells were
depleted from the UCB by means of an immunoabsorbance technique. The
resulting Lin UCB was enriched 21.7-fold ± 38.7-fold
for SP cells relative to the unfractionated UCB (Figure 3A-B;
n = 39; median, 7.7-fold; range,
0.1-fold to 124-fold). The Lin SP cells remained
sensitive to verapamil (Figure 3C), and the verapamil-sensitive gate
represented 1.02% ± 0.47% (n = 6) of the Lin UCB.
For these studies, the analyses of the Lin SP were
restricted to those cells with the dimmest Hoechst staining, which
typically represented 1% of the Lin UCB or less. The
Lin SP was depleted of cells expressing CD2, CD3, CD4,
CD5, CD16, or CD56, and it did not contain cells expressing CD19, CD33,
or CD71 (data not shown).
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| Figure 3.
Hoechst dye efflux by Lin UCB.
The SP cells, as defined by verapamil-sensitive cells, are indicated in
the enclosed box. The data are representative of 39 experiments. (A)
The Hoechst 33342 staining and emission patterns of unfractionated
human UCB. (B) The Hoechst 33342 staining and emission patterns of
Lin UCB in the absence of verapamil. (C) The Hoechst
33342 staining and emission patterns of Lin UCB in the
presence of verapamil.
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The Lin SP contained nearly equivalent proportions of
CD34+ (60.5% ± 23.3%) and CD34
(39.7% ± 23.3%) cells (n = 29; Figure
4A-B). In addition, the Lin
SP was enriched for the brightest CD34+ cells and for
CD34CD38 cells (Figure 4B). When
reanalyzed, the purified CD34Lin SP cells
did not express CD34 even when stained with a pool of monoclonal
antibodies raised against 3 different epitopes of CD34 (QBEnd10,
Immu-133, and Immu-409), indicating that these cells did not merely
express an alternative isoform of CD34 (data not shown). The
CD34+Lin SP cells had additional phenotypic
features typical of primitive progenitors, including low levels of
Thy-1 and HLA-DR expression as well as absent CD45RA and CD71
expression (Figure 4C-F).23,24 In contrast, the
CD34Lin SP cells were predominantly
CD38, HLA-DR, Thy-1, and
CD71 but did express CD45RA (Figure 4C-F). The lack of
expression of CD38, HLA-DR, and Thy-1 on the
CD34Lin SP cells is similar to the
phenotype of the LinCD34 populations
previously reported by Bhatia et al15 and Nakamura et
al.18 By both FACS and microscopic analysis, the
CD34+ and CD34Lin SP cells were
small blast cells with minimal internal complexity and cytoplasm;
however, the CD34Lin SP cells were
distinctly smaller than the CD34+Lin SP cells
(data not shown).
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| Figure 4.
Characterization of the Lin UCB SP for HSC
properties.
(A,B) The expression of CD34 and CD38 on Lin (A) and on
Lin SP UCB cells (B). The SP represented the dimmest
0.5% to 1.0% of the Hoechst-stained Lin UCB cells.
Quadrant statistics are provided for the individual experiment
depicted. (C-F) The expression of Thy-1 (CDw90; C), HLA-DR (D), CD45RA
(E), or CD71 (F) versus that of CD34 within the Lin SP.
In all panels, CD34 is depicted on the x-axis regardless of the
fluorochrome used. Quadrant statistics are provided for the individual
experiments depicted. Each panel is representative of at least 3 experiments. (G,H) Myelo-erythroid hematopoietic progenitors were
quantified from unfractionated UCB, Lin UCB, the
Lin SP, the CD34+Lin SP, and
the CD34Lin SP fractions. These were
enumerated in HPCAs (G) and in 5-week LTC assays on MS5 cells (H).
These data are the mean ± SD of 5 experiments. All but 2 of the
HPCAs detected from CD34Lin SP cells were
derived from cells isolated from a single sort.
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To determine whether myelo-erythroid progenitors were present in either
the CD34+ or the CD34 fractions of the
Lin SP, these populations were isolated and placed into
both short-term HPCAs and LTC assays. To ensure the purity of the cell
preparations, Lin SP cells were first isolated on the
basis of dim Hoechst staining and then restained and re-sorted on the
basis of their expression of CD34. For HPCAs, the Lin UCB
was enriched approximately 80-fold over the unfractionated UCB (Figure
4G). The CD34+Lin SP cells had a clonogenic
frequency similar to that of the Lin UCB cells, whereas
the CD34Lin SP cells were nearly devoid of
progenitors detectable in the HPCA. Similar to the HPCA results, the
Lin UCB and the CD34+Lin SP
cells were both highly enriched for LTC activity, whereas no colonies
were generated from the CD34Lin SP (Figure
4H).
The CD34Lin
SP contains NK-cell progenitors
Despite the observation that the
CD34Lin SP failed to generate progeny in
standard assays of myelo-erythroid progenitors, it remained possible
that this population contained additional progenitors not detectable by
these assays. Because the majority of the
CD34Lin SP cells expressed CD45RA (Figure
4E), an isoform of CD45 common to lymphocytes, we evaluated whether the
CD34Lin SP contained lymphoid progenitors.
In addition to CD45RA, the CD34Lin SP
expressed high levels of CD7 and CD11b (Figure
5), cell-surface markers expressed on
various lymphoid progenitors.25-27 The
CD34Lin SP cells did not express CD10
or CD19 (data not shown), indicating these cells were not obvious early
B-lymphocytes. In addition, the CD34Lin SP
cells did not express the T-lymphocyte-associated antigens CD1a, CD3,
CD4, or CD8 or the mature NK-cell markers CD56 or CD16 (data not
shown). They also did not express CD2 or CD5 (data not shown), antigens
found on very early progenitors of both the T- and the NK-cell
lineages.
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| Figure 5.
CD7 expression by the
CD34Lin SP cells.
The expression of CD34 and CD7 are depicted for Lin UCB
(A) and compared with that of the Lin SP (B).
Co-expression of CD7 with CD45RA (C) and CD11b (D) is demonstrated
within the Lin SP. The SP represented the dimmest 0.5%
to 1.0% of the Hoechst-stained Lin UCB cells. Quadrant
statistics are provided for the individual experiments depicted. Each
panel is representative of at least 3 experiments.
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Because the CD7+CD34Lin SP
cells expressed markers found on lymphoid cells but did not obviously
fit into current lymphoid developmental schemas, we tested whether
these cells could generate lymphoid progeny in vitro. In preliminary
studies, CD34Lin SP cells were placed in
suspension cultures that support the growth of relatively mature
lymphoid progenitors.28 Whereas Lin UCB and
CD34+Lin SP cells grew under some of these
conditions, none of the conditions supported the growth of
CD34Lin SP cells (data not shown). This
observation, coupled with the absence of markers found on mature
lymphoid cells, suggested that the
CD7+CD34Lin SP cells were not
mature lymphoid cells.
To determine whether the
CD7+CD34Lin SP contained more
primitive lymphoid progenitors, these cells were cultured on stroma using modifications of previously described in vitro culture systems that support the growth of primitive NK-cell progenitors (Table 1,
conditions A-D).29-31 All of these conditions except
condition B contained IL-15, a cytokine known to enhance the maturation of NK cells. As previously described, the stromal cells or condition B
may provide factors that replace the effects of IL-15 on NK-cell development.29-31 In our studies, Lin SP
cells were isolated and then re-sorted into CD7+ and
CD34+ subfractions. CD34+ cells, as well as
cells expressing CD2, CD3, CD5, and CD56, were stringently excluded
from the CD7+CD34Lin SP
subfraction to ensure that CD34+ HSCs and committed
lymphoid progenitors would not contribute to the culture results
(Figure 6). After 6 to 8 weeks of growth, the stroma-based cultures were assessed for total cell expansion and
for the proportion of cells expressing CD56+ and other
NK-cell markers (Figure 6; Table 4). In
all conditions tested, both the CD34+Lin SP
and the CD7+CD34Lin SP
fractions proliferated. However, both purified cell fractions exhibited
wide ranges in their expansion under each of the culture conditions
tested (Table 4). Under culture condition C, the
CD7+CD34Lin SP cells expanded
approximately 3-fold more than the CD34+Lin
SP fraction. Under all of the conditions tested, the
CD7+Lin SP consistently gave rise to higher
proportions of CD56+ progeny than were derived from the
CD34+Lin SP cells, and under condition C, the
increased proportion of CD56+ cells derived from the
CD7+Lin SP was statistically significant
(P < .05, Table 4). This finding argues that the
generation of CD56+ cells from the
CD7+Lin SP was not due to contamination with
CD34+Lin SP cells.
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| Figure 6.
Characterization of NK cells derived from
Lin SP cells.
Lin SP cells were isolated and re-sorted on the basis of
their expression CD7 or CD34 (B). The Lin SP resolved 2 distinct subpopulations when compared with Lin UCB (A).
The reanalysis of sorted CD7+ and CD34+ SP
cells (C,D, respectively) consistently demonstrated the purity of the
fractions. The CD7+CD34Lin SP
cells and CD34+Lin SP cells were then grown
in the different stroma-based cultures described in Table 1. The
immunophenotype of the progeny of
CD7+CD34Lin SP cells (E, G, I)
and CD34+Lin SP cells (F, H, J) grown under
culture condition D (Table 1) are shown. For each culture, one quarter
of the human CD45+ progeny were used to confirm the
presence and maturity of CD56+ cells by their
immunophenotype. Quadrant statistics are provided for the individual
experiments depicted. These data are representative of 4 cultures
derived from CD7+Lin SP cells and 2 cultures
of CD34+Lin SP cells. An additional 2 cultures of CD34+Lin SP expanded but did not
yield CD56+ cells after 8 weeks in culture.
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Under culture conditions A, B, or C, either the total cell expansion or
the degree of maturation of the progeny was insufficient to perform
NK-cell functional assays. In order to more effectively generate mature
NK cells, a fourth culture condition, condition D, was designed.
Condition D included a 2-week "initiation" phase in the presence of
KL, F3L, IL-2, IL-7, and IL-15. This was followed by a 4-week
"expansion" phase in which the media were supplemented with only KL
and IL-15, and finally by a 2-week "maturation" phase in which the
media contained KL, IL-2, and IL-15. In 4 of 6 experiments, the
CD34+Lin SP cells expanded under these
conditions (Table 4), and in 2 of these 4 experiments,
CD56+ progeny were detected (Table 4; Figure 6). The
CD7+CD34Lin SP cells expanded
in 4 of 6 experiments under condition D, and CD56+ progeny
were detected in all of these cultures (Table 4; Figure 6). In
condition D, 28.1% ± 6.5% of the total cells generated from the
CD7+CD34Lin SP fraction had the
CD56+CD3CD94+ phenotype,
indicating that these were mature NK cells.29-31 The remainder of the cells had phenotypes more consistent with those of
immature NK cells or non-NK cells (Figure 6). To confirm that the cells
generated from the CD7+CD34Lin
SP under condition D were NK cells, the cultured cells were tested for
their ability to lyse NK-sensitive target cells by means of a newly
developed FACS assay for NK-cell activity (Figure
7). In 2 of 3 cultures assayed, lysis of
the NK-sensitive cell line K562 was readily observed, whereas the
NK-insensitive cell line Raji was not lysed (Figure 7C). K562 lysis was
observed even at relatively low effector-to-target ratios, suggesting
that a large proportion of the cultured cells possessed NK functional
activity.
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| Figure 7.
NK cytotoxic activity in the progeny of
CD7+CD34Lin SP cells.
Panels A and B depict 7AAD uptake by CFSE-labeled K562 cells when
cultured 4 hours without effectors (A) as compared with those cultured
4 hours with putative NK cells generated from
CD7+CD34Lin SP under condition
D (B). Panel C depicts the percentage of cell lysis for K562 or Raji
target cells at various effector-to-target ratios. The effector cells
were putative NK cells derived from
CD7+CD34Lin SP cells grown
under condition D (Table 1). These data are representative of 2 cultures (of 3 tested) where lysis of K562 target cells was
clear.
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Discussion |
In the current study, we found that human UCB SP cells were
largely CD34, similar to SP cells derived from murine,
rhesus, and human bone marrow. In contrast to the rhesus bone marrow
SP, up to 50% of the CD34 UCB SP expressed markers found
on mature NK cells and T cells. This finding was not surprising since
some human T and NK cells can express high levels of MDR activity that
could result in the efflux of the Hoechst dye.32 The
differences in the content of the human UCB SP and the rhesus bone
marrow SP could be due to technical differences in the Hoechst staining
and analysis procedures, differences between bone marrow and UCB, or
differences between species. The depletion of cells expressing
lineage-commitment markers eliminated the contaminating mature
lymphocytes from the UCB SP. The resulting Lin UCB SP was
composed of distinct CD34+ and CD34
populations. The phenotype and in vitro behavior of the
CD34+Lin SP cells was consistent with that of
primitive multipotent human hematopoietic progenitors previously
described by a number of groups.4-6,23,33 The
CD34+Lin SP cells were small blast cells with
high levels of CD34 expression, dim to undetectable levels of CD38 and
Thy-1 expression, intermediate levels of HLA-DR expression, and
undetectable levels of CD33, CD45RA, and CD71 expression. These cells
were enriched for myelo-erythroid progenitors in both short-term and
5-week long-term colony-forming unit assays and gave rise to NK cells
on stroma supplemented with various cytokines. While the
CD34+Lin SP cells were not more enriched in
short-term or 5-week long-term colony-forming units than
Lin UCB (Figure 4), the culture conditions used in these
studies were not optimized for primitive progenitors. Future studies
using more extended LTCs or other conditions designed to support the growth of very primitive hematopoietic progenitors will be useful for
determining whether the SP fractionation procedure segregates primitive
CD34+ progenitors from more mature progenitors within the
Lin fraction.
In contrast to the CD34+Lin SP cells, the
CD34Lin SP cells failed to grow in standard
short-term or long-term myelo-erythroid colony-forming unit assays. The
majority of the CD34Lin SP cells
coexpressed CD7, CD45RA, and CD11b, yet failed to express CD38 or a
variety of lineage-commitment markers, including CD1a, CD2, CD3, CD4,
CD5, CD8, CD10, CD16, CD19, CD33, CD56, CD71, HLA-DR, and Thy-1. This
suggested that the CD34Lin SP cells may
have been an early lymphoid progenitor population. In support of this,
the CD7+CD34Lin SP fraction
readily expanded into NK cells on stroma supplemented with cytokines,
indicating that the CD7+CD34Lin
SP contained primitive NK progenitors. A similar
CD7+CD34Lin population has not
been previously described, and this cell population cannot be easily
placed into current models of lymphocyte ontogeny. NK progenitors
isolated from fetal liver are CD7brightCD34
but are also typically more than 95%
CD38+CD56+.25 Of the T-cell and
NK-cell progenitors described to date, the
CD7+CD34Lin SP cell is
phenotypically most similar to a putative shared T-cell and NK-cell
progenitor that was cloned from fetal liver.28 This cell
is
CD7brightCD11b+CD1CD3CD4CD8CD56;
however, unlike the CD7+CD34 Lin SP cells,
the fetal liver cells express CD2 and HLA-DR and respond to IL-2.
Similarly, the CD7+Lin SP cells are not
obvious T-cell progenitors. Most primitive prethymic T-cell progenitors
are derived from CD34+CD7dim cells detectable
in either bone marrow or fetal liver,27,30 and fetal
thymic T-cell progenitors generally coexpress CD38, CD2, CD5, CD1a, or
CD3 during their development.25 On the basis of these
prior observations, it remains unclear whether the
CD7+CD34Lin SP is a previously
unreported population of committed NK-cell progenitors or whether this
population represents a novel multipotent cell whose full activity was
not detected in the assays used in this report. Detecting
myelo-erythroid, B-cell, T-cell, and dendritic-cell growth from the
CD7+CD34Lin SP may require
different assays, different culture conditions, or longer culture
durations than those used in this report. Additional studies using both
in vitro and in vivo models for T-cell, B-cell and myelo-erythroid
growth are ongoing to further delineate the full developmental
potential of the CD7+CD34Lin SP cells.
In addition to the CD34+Lin SP and
CD7+CD34Lin SP fractions, a
small population of
CD34CD7Lin SP cells were
routinely detected (Figure 5B, lower left quadrant |
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Abstract
Introduction