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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4145-4151
In Vitro Identification of Single
CD34+CD38 Cells With Both Lymphoid and
Myeloid Potential
By
Qian-Lin Hao,
Elzbieta M. Smogorzewska,
Lora W. Barsky, and
Gay M. Crooks
From the Division of Research Immunology/Bone Marrow Transplantation,
Childrens Hospital Los Angeles, Los Angeles, CA.
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ABSTRACT |
Human hematopoietic stem cells are pluripotent, ie, capable of
producing both lymphoid and myeloid progeny, and are therefore used for
transplantation and gene therapy. An in vitro culture system was
developed to study the multi-lineage developmental potential of a
candidate human hematopoietic stem cell population, CD34+CD38 cells.
CD34+CD38 cells cocultivated on the murine
stromal line S17 generated predominantly CD19+ B-cell
progenitors. Transfer of cells from S17 stroma to myeloid-specific conditions ("switch culture") showed that a fraction of the
immunophenotypically uncommitted CD19 cells generated on
S17 stroma had myeloid potential (defined by expression of CD33 and
generation of colony-forming unit-cells). Using the switch culture
system, single CD34+CD38 cells were
assessed for their lymphoid and myeloid potential. Nineteen of 50 (38%) clones generated from single
CD34+CD38 cells possessed both B-lymphoid
and myeloid potential. 94.7% of the
CD34+CD38 cells with lympho-myeloid
potential were late-proliferating (clonal appearance after 30 days),
demonstrating that pluripotentiality is detected significantly more
often in quiescent progenitors than in cytokine-responsive cells
(P = .00002). The S17/switch culture system permits the in
vitro assessment of the pluripotentiality of single human hematopoietic
cells.
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INTRODUCTION |
HEMATOPOIETIC STEM CELLS (HSC) have the
unique capacity to provide long-term and complete restoration of
myelopoiesis and lymphopoiesis after marrow ablation. The clinical
fields of hematopoietic cell transplantation and gene therapy have
stimulated intense interest in ex vivo manipulation of HSC. However,
conclusions about the effects of ex vivo manipulations have been based
on in vitro assays of committed progenitors. These in vitro assays may
be misleading when used as surrogate markers for true pluripotent HSC.1 An assay able to detect single cells possessing both myeloid and lymphoid potential (pluripotentiality) is required to
understand how approaches aimed at HSC proliferation may affect HSC
function.
In vivo assays of human hematopoiesis have been developed in which
human hematopoietic cells engraft in immunodeficient
mice.2-4 These models have demonstrated the existence of
human pluripotent cells either by limiting dilution analysis or by
clonal integration of a retroviral marker gene.5,6 Until
recently, in vitro systems of human hematopoiesis have been limited to
lineage-specific cultures.7-12 Our goal in the present
studies was to determine whether lineage-specific culture systems for
myeloid and lymphoid cells could be modified to show in vitro the
presence of pluripotent human hematopoietic cells. In this report we
describe the development of a switch culture system based on
cocultivation on the murine stromal line S17 in which single cord blood
CD34+CD38 cells with pluripotentiality can
be identified. Furthermore, the lympho-myeloid cells so detected are
found almost exclusively in late-proliferating
CD34+CD38 cells, demonstrating that more
rapidly proliferating, cytokine-responsive progenitors lack
multilineage potential.
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MATERIALS AND METHODS |
Isolation of CD34+CD38 cells.
Umbilical cord blood samples were collected by the Labor and Delivery
Staff at Kaiser Permanente Hospital Sunset (Los Angeles, CA) following
clamping of the cord as previously described9 and according
to guidelines reviewed by the Committee on Clinical Investigations at
Childrens Hospital Los Angeles. Mononuclear cells were prepared using
Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density centrifugation
within 24 hours of collection. Enrichment for CD34+ cells
was performed as per manufacturer's guidelines using the MiniMACS
system (Miltenyi Biotec, Auburn, CA). CD34+ enriched cells
were washed in phosphate-buffered solution (PBS; Irvine Scientific,
Santa Ana, CA) and incubated for 30 minutes at 4°C in fluorescein
isothiocyanate (FITC)-CD34 (HPCA2; Becton Dickinson Immunocytometry
Systems [BDIS], San Jose, CA) and phycoerythrin (PE)-CD38 (leu 17;
BDIS). CD34+CD38 cells were then isolated
using the gating previously described9 on a FACSVantage
flow cytometer (BDIS) with an argon laser tuned at 488 nm using Lysys
II software (BDIS). CD34+CD38 cells, defined
by the R2 gate in Fig 1, comprised
3.53% ± 0.68% of the CD34+ cells.
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| Fig 1.
CD34+CD38 cord blood cells
are immunophenotypically uncommitted to lymphoid or myeloid lineages.
Three-color analysis of CD34+ enriched cord blood cells
from region R1 (based on forward and side scatter (not
shown9). (B) Region R2 defining
CD34+CD38 cells (ie, high CD34 expression
with CD38 expression less than half the maximal fluorescence of the
isotype control). R2 comprises 3.53% ± 0.68% (mean ± SEM) of
all CD34+ cells (defined by upper and lower right
quadrants). (C, D, and E) The lack of expression of lineage-specific
antigens on cells from R2. (A and F) Respective isotype controls.
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An aliquot of CD34+ MiniMACS enriched cells was removed for
analysis of lineage-specific antigen expression using three-color fluorescence-activated cell sorter (FACS) analysis. For these studies
CD34+ enriched cells were incubated with FITC-CD34,
APC-CD38 (leu 17; BDIS), and one or more of the following antibodies
conjugated to PE: CD19 (leu 12; BDIS), CD10 (Calla; Coulter, Hialeah,
FL), CD33 (My 9; Coulter), CD11b (leu 15; BDIS), and CD14 (My 4;
Coulter). Analysis was performed on the FACSVantage using argon and
HeNe lasers and CellQuest software (BDIS).
Stromal cultures.
S17 stroma is a murine bone marrow (BM) stromal line and was generously
provided by Dr Kenneth Dorshkind (University of California at Los
Angeles).13 S17 stroma was expanded in RPMI 1640 (Irvine Scientific), 5% fetal calf serum (FCS), 50 µmol/L 2-mercaptoethanol (2-ME; Sigma, St Louis, MO), penicillin/streptamycin (P/S; Gemini Bio
Products, Calabasas, CA) and glutamine (Gemini Bio Products). S17
stroma was not irradiated before plating of human hematopoietic cells
and was maintained in a confluent state during long-term culture by
contact inhibition. Primary human stroma (HS) was prepared from
mononuclear BM cells as previously described.9,10 HS was
then trypsinized, irradiated at 20 Gy, and replated at a concentration of 7 × 103 cells/well in 96-well plates (Becton Dickinson
Labware, Lincoln Park, NJ).
Primary cultures.
Primary cultures consisted of cocultivation of
CD34+CD38 cells on S17 in "lymphoid
medium" (RPMI, 5% FCS, 2-ME, P/S, glutamine). In some experiments
(see Fig 6) primary cultures were established on either S17 or HS with
either lymphoid medium or "myeloid medium-noHC/GF" (ie, myeloid
medium [see below] without hydrocortisone or added growth factors).
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| Fig 6.
B-cell potential is lost during primary culture on HS.
CD34+CD38 cord blood cells were cultured
for 1 week in each of the four primary culture conditions shown and
then switched to BSC on S17 stroma to measure maintenance of B-cell
potential. At days 34 and 60 after switching, cells were obtained from
BSC, counted, and analyzed by FACS for CD19 expression. Bars shown are
means (±SEM) compiled from three independent experiments. LM,
lymphoid medium; MM, myeloid medium-noHC/GF (see Materials and
Methods). ( ), HS/LM; ( ), HS/MM; ( ), S17/LM; ( ), S17/MM.
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Immunophenotypic analysis of cultured cells.
Cells from primary cultures were incubated first with 1% human IVIG
(Cutter, Berkely, CA) to block nonspecific binding, and then with
PE-CD33 and FITC-CD19. In view of the nonspecific background staining
which occurs in cultured cells, it was necessary to exclude surface
antigen expression occurring at low frequency (<5%) from the
analyses. Thus, the presence of myeloid cells in primary culture was
defined as CD33 expression in greater than 5% of cells. B-lymphoid differentiation was defined as CD19 expression in greater than 20% of
cells.
Lineage-specific (switch) cultures.
Hematopoietic cells were switched from primary culture into either
B-cell-specific culture ("BSC," ie, S17 and lymphoid medium as
described above) or myeloid-specific culture ("MSC," ie, HS and
myeloid medium = Iscoves Modified Dulbecco's Medium [IMDM; GIBCO-BRL, Bethesda, MD], 30% FCS, 1% bovine serum albumin [BSA; Sigma], 2-ME, P/S, glutamine, 106 mol/L hydrocortisone
[Sigma] and the myeloid growth factors interleukin-3 [IL-3; 10 ng/mL], IL-6 [50 U/mL], and Steel factor [SF, 50 ng/mL]).9,10 Hydrocortisone was included in MSC to prevent
B-cell proliferation; cytokines were added to induce myeloid
differentiation. The presence of myeloid progenitors in MSC was assayed
by replating nonadherent cells into semisolid medium (myeloid medium
with the addition of 1.3% methylcellulose, 50 ng/mL
granulocyte-macrophage colony-stimulating factor [GM-CSF] and 2 U/mL
erythropoietin) and enumeration of colony-forming unit-cells (CFU-C)
after 14 days.
Cloning efficiency of CD34+CD38
cells grown on S17 stroma.
Cloning efficiency was determined by plating
CD34+CD38 cells by the Automated Cell
Deposition Unit (ACDU) of the FACSVantage onto established S17 stroma
either in limiting dilution or as single cells into each well of
96-well plates. Accuracy of ACDU sorts was checked by sorting single
chicken red blood cells (Axell; Accurate Chemical and Scientific Corp,
Westbury, NY) and FITC-conjugated beads (Calibrite beads; BDIS) into
each well of 96-well flat-bottom microtiter plates (n = 6). These
checks showed that an average 6% of wells contained no visible cells.
No instances of more than one cell per well were seen. In
the limiting dilution experiments, between 22 and 144 wells containing
each of the following cell numbers were plated: 1, 3, 10, 20, 30, 40, 80 cells. Timing of clonal appearance was estimated only in the
experiments using single-cell deposition. Wells were inspected every
week and the presence of new clones recorded to note the timing of
first appearance. Because B cells are small and grow as a dispersed
population rather than as easily visible cobblestone areas, only clones
containing more than 500 cells could be visualized with certainty and
were thus scored as positive. Clones containing more than 1,000 cells were procured for cell enumeration after Trypan blue staining. Those
clones that were visible but too small to enumerate were recorded as
containing 500 to 1,000 cells.
Clones were obtained for CD19 staining, FACS sorting, and switch
culture when they contained at least 3,000 cells. This occurred within
1 week of first identification and was constant for both late and early
appearing clones large enough for analysis. In single-cell studies,
myeloid potential within the CD19 cells of each clone
was defined as a combination of (1) proliferation of cells after
switching CD19 cells to MSC, and (2) CD33 expression in
greater than 5% of cells and/or CFU-C production after
switching to MSC.
Statistical analysis.
Significant differences between data groups were determined by
T-test using Fisher's Exact and Aspin-Welch methods.
Cloning efficiency using limiting dilution analysis (LDA)
data was analyzed by Poisson statistics and the weighted mean method.
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RESULTS |
Our goal in these studies was to determine whether single human
hematopoietic cells with both lymphoid and myeloid potential (pluripotentiality) could be detected in vitro.
CD34+CD38 cells lack expression
of lineage-specific antigens.
We sought to initiate cultures with a CD34+ subpopulation
which lacked immunophenotypic evidence of lineage commitment.
Expression of lineage-specific antigens on CD34+ subsets
(defined by CD38 expression) from cord blood was studied using
three-color FACS analysis. The CD34+CD38
immunophenotype defines a highly primitive, largely quiescent population of hematopoietic progenitors.9,10,14-16 Only
cells defined as CD34+CD38 using the
stringent R2 gate (ie, high CD34 expression with CD38 expression less
than half the maximum fluorescence of the isotype control, Fig 1)
uniformly lacked expression of the B-cell antigens (CD19 and CD10) and
the myeloid antigens (CD33, CD14, and CD11b). Therefore, we used the R2
gate to define CD34+CD38 cells for all
studies to maximally enrich for uncommitted cord blood cells.
Cloning efficiency of B-lymphoid progenitors from single cord blood
CD34+CD38 cells.
Cocultivation of human CD34+CD38 cells on
S17 stroma has recently been shown to generate cultures containing
predominantly B-lymphoid progenitors; 80% to 95% of cultured cells
express CD19, CD10, and CD45, do not express CD34 or CD20, and are
predominantly germline at the Ig locus.12 To ascertain the
feasibility of adapting the S17 system to analysis of single cells, the
cloning efficiency of B-lymphoid progenitors from cord blood
CD34+CD38 cells was determined.
CD34+CD38 cells were isolated by FACS and
plated onto S17 stroma in limiting dilution. Day 40 cloning efficiency
of cord blood CD34+CD38 cells was 2.4% ± 0.4% (mean ± SEM, n = 3). In experiments with single-cell
plating by ACDU, cloning efficiency of
CD34+CD38 cord blood cells was 3.7% ± 1.3% (n = 3). Combining these data, the frequency of
CD34+CD38 cells capable of proliferating on
S17 stroma by day 40 was 3.02 ± 0.67% (n = 6).
Immunophenotype of clones generated from single
CD34+CD38 cells on S17 stroma.
Of 224 clones generated from single
CD34+CD38 cells on S17 stroma (n = 4), 88 clones contained sufficient cells to be analyzed for expression of the
B-cell-specific antigen CD19 by FACS. The unambiguous presence of
B-cell progenitors (defined as >20%
CD33CD19+ cells) was found in 85 of 88 (96.6%) clones (Fig 2).
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| Fig 2.
CD19 expression in clones from single
CD34+CD38 cells in S17 culture. Bars
indicate mean ± SEM from four individual experiments (total 88 clones).
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Although most clones contained predominantly CD19+ cells,
CD19 cells were also present, and comprised 5% to 80%
of the cells. Immunophenotypic evidence of myeloid differentiation
(defined as CD33 expression in >5% of cells) was not detectable in
any clones. Analysis of additional clones showed no expression of another myeloid antigen, CD11b.
Onset of clonal proliferation and clone size.
The timing of clonal proliferation was determined by plating single
cells in individual wells and noting the time at which clones first
became visible during 100 days of culture. The time of appearance of
the clones generated from single CD34+CD38
cells was variable, ranging from as early as day 13 to as late as day
76. Most clones appeared before day 30 (from "early
proliferating" cells). However, "late-proliferating cells"
(those which formed clones after day 30) comprised 1.6% (34 of 2,112)
of all CD34+CD38 cells plated and 26.2% (34 of 130) of all clones (Fig 3A). The relationship between timing of clonal proliferation and generative capacity (clonal size) was also determined. Late-proliferating cells
produced clones containing a significantly greater number of progeny
(1.77 ± 0.33 × 104 cells, mean ± SEM) than did
early proliferating cells (0.54 ± 0.11 × 104 cells)
(P < .01) (Fig 3B).
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| Fig 3.
Proliferation from single
CD34+CD38 cord blood cells. (A) Timing of
clonal appearance. Above the bars are shown the number of clones
analyzed at each time point. (B) Relationship of timing of clonal
appearance to generative capacity. Bars denote mean number of
cells/clone ± SEM. Data for (A) and (B) are compiled from a total 130 clones initiated from 2,112 single
CD34+CD38 cells.
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CD19 cells from primary S17 cultures have
lymphoid and myeloid potential.
Having established that CD34+CD38 cells are
preferentially directed to B-lymphoid differentiation on S17 stroma
without immunophenotypic evidence of myeloid commitment, we next
determined if the functional capacity to differentiate into the myeloid
lineage was preserved on S17. A switch culture system in which cells
from primary B-lymphoid cultures were replated into MSC was used to
reveal the presence or absence of myeloid progenitors in primary S17
cultures. To determine the lineage potential of the minority of cells
not yet committed to B-lymphoid differentiation, CD19
cells were separated by FACS from the more numerous CD19+
cells from day 14 to day 60 of primary S17 culture (n = 3).
CD19 and CD19+ populations were then split
and each replated into BSC and MSC (Fig 4).
CD19 cells replated into BSC proliferated with a 20- to
50-fold increase in cell number (n = 3). CD19 antigen was expressed on 44% to 88% of the cells generated from CD19 cells
in BSC. CD19 cells isolated after at least 5 weeks of
primary S17 culture retained the ability to differentiate into
CD19+ B-lymphoid progenitors in BSC.
CD19 cells also differentiated into myeloid cells when
cultured in MSC. After an initial 3 weeks on S17 stroma, isolated
CD19 cells replated into MSC expanded from 113- to
1,600-fold and cell proliferation persisted for over 6 weeks (n = 3);
CD33 expression was present in over 50% of cells. To confirm the
lineage commitment of proliferating cells functionally, nonadherent
cells from MSC were replated into methylcellulose culture to detect
myeloid progenitors. 0.67% ± 0.24% (mean ± SEM) of
CD19 cells gave rise to CFU-C and these CFU-C could be
detected in CD19 populations isolated after at least 60 days in S17 culture. Long-term culture-initiating cells
(LTC-IC)8 were detected in CD19 populations
after an initial 35 days of S17 culture. Thus, the CD19
fraction of S17 primary cultures generates two populations of progenitors: B-lymphoid progenitors that express CD19 after secondary culture with S17 and myeloid progenitors revealed only when they are
induced to proliferate and differentiate in myeloid-specific conditions.
CD19+ cells are committed to B-cell differentiation.
When CD19+ cells from primary S17 cultures were isolated by
FACS and replated in BSC, they underwent no further proliferation but
were maintained viably for at least 4 weeks. After switching to MSC
(which contains hydrocortisone), CD19+ cells showed no
expansion and disappeared completely within 5 days. Thus,
CD19+ cells in S17 culture were irreversibly committed to
B-cell differentiation and did not display myeloid potential under the
conditions described here.
Single cord blood CD34+CD38
cells have both myeloid and lymphoid potential.
The S17 switch culture system was next applied to single
CD34+CD38 cells to conclusively prove
their pluripotentiality. Clones expanded from single
CD34+CD38 cord blood cells were obtained
from S17 and incubated with CD33 and CD19; CD19 cells
were isolated from each clone and switched into MSC.
Of 2,112 single CD34+CD38 cells plated in
individual wells, a total of 50 clones contained sufficient cells for
FACS analysis and switch culture. Of these 50 clones, 31 showed only
B-lymphoid potential, ie, they contained greater than 20%
CD19+ cells, but showed no myeloid potential upon switching
of the CD19 cells to MSC. CD19 cells
comprised 35.9% ± 3.5% of the total cells in the 50 clones analyzed. Nineteen clones (38% of all clones analyzed) showed both
B-lymphoid (CD19 expression) and myeloid potential (in MSC), representing 0.9% of all CD34+CD38 cord
blood cells plated. In the 19 bipotent clones, the CD19
cells comprised 51.2% ± 5.3% of the total cells in each clone. Of
the 50 clones analyzed, 22 appeared before day 30, but only one clone
(4.5%) which appeared at day 20 demonstrated both B-lymphoid and
myeloid potential (Fig 5). Of the 28 clones
from late-proliferating cells, 18 clones (64.3%) showed both
B-lymphoid and myeloid potential. Thus, pluripotent cells were found
almost exclusively in the subpopulation of
CD34+CD38 cells which proliferate late in
culture (P = .00002, early v late pluripotent cells).
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| Fig 5.
Relationship of B-lymphoid/myeloid potential with timing
of proliferation. Shown on the vertical axis is the percent clones with
both B-lymphoid and myeloid potential over the total clones analyzed at
each time point. Raw numbers of bipotent clones over total clones
analyzed are shown above each bar. Data are compiled from a total of
2,112 CD34+CD38 cells plated as single
cells per well onto primary S17 culture and analyzed by FACS and switch
culture.
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B-cell potential is rapidly lost during cocultivation on primary HS.
Cocultivation of human cells on primary HS has become the standard in
vitro assay of human myelopoiesis.7,17 Therefore, we
determined whether the switch culture system could be adapted to use HS
instead of S17 stroma during the primary culture of CD34+CD38 cells. To determine if B-cell
progenitors can be maintained on primary HS, cells were cultured
initially in bulk on HS and then switched to BSC to assay B-cell
potential. CD34+CD38 cells cultured in bulk
for 1 week on HS could generate B-cell progenitors after switching to
BSC, although at reduced numbers compared with cells cultured on S17
stroma continuously (n = 3, Fig 6). The
superiority of S17 over HS in maintaining B-cell progenitors was seen
irrespective of whether primary cultures contained lymphoid medium or a
modification of myeloid medium (ie, myeloid medium without
hydrocortisone or growth factors). Thus, the critical component
required for maintenance of B-lymphoid potential is the presence of S17
stroma.
Cultures were also initiated with single
CD34+CD38 cord blood cells plated in primary
culture onto HS (and lymphoid medium). Resultant clones were switched
to BSC and analyzed by FACS for evidence of lymphoid development. From
a total 607 CD34+CD38 cells, 47 clones
developed and 7 were large enough for FACS analysis. B cell progenitors
(CD19+CD33 cells) were absent in all clones
studied. All clones showed myeloid differentiation
(CD33+ > 10%). Thus, although S17 and HS are both able
to maintain primitive myeloid progenitors for prolonged periods, B-cell
progenitors are rapidly lost during culture on HS.
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DISCUSSION |
In this report we have developed a switch culture assay to demonstrate
the presence of human progenitors with both lymphoid and myeloid
potential. Single CD34+CD38 cells from cord
blood are plated initially onto the murine stromal line S17 to allow
clonal proliferation. In the primary S17 cultures proliferation gives
rise to two populations: cells irreversibly committed to B-lymphoid
differentiation (CD19+ cells) and a more primitive
CD19 population, which is yet to express either myeloid
or lymphoid antigens but has the potential to differentiate into either
or both lineages. The existence of hematopoietic progenitors with the
capacity for B-lymphoid and myeloid differentiation but which lack the
potential for T-lymphoid development cannot be excluded by these
studies. Application of a T-lymphocyte culture system to the
single-cell switch culture assay described here will delineate further
the differentiation pathways of uncommitted hematopoietic stem cells.
The presence of S17 in primary culture was crucial for the switch
culture system when applied to single cells. Use of primary human BM
stroma in otherwise identical culture conditions did not permit clonal
expansion into B-lymphoid cells. The reason for this lineage
restriction is unclear, but presumably elements within HS either
prevent lymphoid commitment from pluripotent cells and/or
inhibit the expansion of lymphoid progeny to detectable numbers.
The S17 stromal line was initially identified by its ability to support
both murine myelopoiesis and B lymphopoiesis,13 and has
been shown to be superior to primary murine BM stroma and numerous
other murine stromal lines in its ability to support murine long-term
repopulating cells in vitro.18,19 Studies of human
hematopoiesis have recently reported that S17 stroma permits B-lymphoid
differentiation from cord blood CD34+ and
CD34+CD38 cells into cultures containing
80% to 95% CD19+ cells.11,12 This current
report shows that S17 stroma also maintains primitive human
hematopoietic progenitor cells and that the myeloid potential of these
cells can be revealed by switching to conditions allowing myeloid
differentiation.
The ability of other murine stromal lines to support human myelopoiesis
and B lymphopoiesis has been reported recently.20,21 The
Sys1 murine BM stromal line allowed the proliferation of human cord
blood CD34hi/lin cells into myeloid and
B-lymphoid lineages.20 The MS5 BM stromal line supported
LTC-IC from human BM and has been recently used to identify
B-lymphoid/myeloid progenitors from cord blood
CD34+CD38low cells (defined as 25% of
CD34+ cells with the lowest CD38
expression).21,22 Both of these studies identified the
presence of B-lymphoid and myeloid cells by FACS analysis without
functional confirmation of lineage commitment and used limiting
dilution analysis for assessment of single-cell behavior.
In the present report with S17 stroma, the high percentage of cells
expressing CD19 in each clone made determination of the presence of
B-lymphoid cells unambiguous. CD19+ cells were irreversibly
committed to B-lymphoid lineage; they had lost CD34 expression, died
rapidly in myeloid culture, but were sustained in B-cell culture.
However, expression of myeloid antigens (CD33 and CD11b) was never
found in more than 5% of cells from each clone. Detection of
lineage-specific antigens below this level is difficult in cultured
cells because of the nonspecific staining commonly seen. Thus, we chose
to base the definition of myeloid potential on functional analysis, ie,
proliferation in myeloid culture and production of clonogenic myeloid
progenitors. Because the S17 culture system allows B-lymphoid
differentiation from single CD34+CD38 cells
with concomitant maintenance of functionally primitive myeloid
progenitors with great proliferative capacity, myeloid potential could
be confirmed functionally and unequivocally after serial replating of
CD19 cells into myeloid stromal and methylcellulose
cultures. Furthermore, the generative capacity of the B-cell and
myeloid progenitors upon replating of the CD19 cells
allow further study of each population after further cell expansion.
In attempting to define pluripotent cells, it is important that assays
are initiated with as homogeneous and uncommitted population as
possible. When total CD34+ cord blood cells are
cocultivated on S17 stroma, definite CD33 expression is seen early in
bulk cultures11 and in clones from early proliferating
cells plated in limiting dilution (unpublished data, April
1997). These CD33+ cells (which do not produce
CFU-C upon replating) are presumably derived from committed myeloid
progenitors sufficiently differentiated at the time of plating to
express CD33. Thus, there is a potential for contamination by committed
myeloid progenitors when clones are obtained by limiting dilution using
heterogeneous starting populations. The stringent definition of
CD34+CD38 cells described by the R2 region
(which excludes all but the 3% to 4% of CD34+ cells with
the highest CD34 and lowest CD38 expression) was therefore used in
these studies.
The frequency of B lympho-myeloid progenitors detected using the S17
culture system (0.9% of CD34+CD38 cells) is
lower than that reported recently by Berardi et al,21 who
found that 7% of CD34+CD38low cells plated by
limiting dilution onto MS5 stroma gave rise to cultures with
simultaneous CD19 and CD11b expression.The higher frequency may be due
to differences in the population of cells used to initiate the
cultures, the use of limiting dilution analysis, or functional
differences in the supporting stromal lines MS5 and S17. It is also
possible that both this study and our own underestimate the true
frequency of pluripotent cells as the sensitivity of such analyses
depends on sufficient clonal growth. In vivo studies of human B
lympho-myeloid cells in nonobese diabetic/severe combined
immunodeficient (NOD/SCID) mice have found that 0.16% of
CD34+CD38 cord blood cells are
SCID-repopulating cells that can generate both B-lymphoid and myeloid
cells.6 The comparatively low frequency of bipotent cells
in these in vivo studies may be secondary to low seeding efficiency in
the NOD/SCID mouse. A seeding efficiency of 10% to 20%, similar to
that of CFU-S in murine transplant models,23 would result
in an estimate of the frequency of pluripotent HSC similar to that
presented in this report.
Several in vivo murine transplant studies have led to the notion of a
hierarchy of stem cells/progenitors based on quiescence. Long-term
repopulating cells can be distinguished from more mature progenitors
such as CFU-S by their resistance to cell-cycle-dependent agents.24-27 In vitro murine studies using the cobblestone
area forming cell (CAFC) assay showed that cell populations that
proliferated late in culture (day 28 CAFC) were those which contributed
most to long-term repopulation.28,29 Our previous studies
using human myeloid long-term cultures have shown the functional
heterogeneity of the CD34+CD38 population in
cord blood and in BM.9,10 Although most
CD34+CD38 cells which proliferate in myeloid
conditions do so during the first 30 days of culture, a small
proportion (termed the extended LTC-IC) form clones late in culture,
ie, after 30 to 80 days. CD34+CD38 cells
that proliferate late in myeloid culture have a greater generative
capacity than early proliferating cells.10 These and other
in vitro studies using human hematopoietic cells support the concept
that quiescence or "cytokine-nonresponsiveness" is associated
with the most primitive human hematopoietic
progenitors.30-32 The same relationship between late
proliferation and greater generative capacity has been noted in this
report using CD34+CD38 cells grown on S17
stroma in B-lymphoid conditions. The characteristic of
pluripotentiality, fundamental to HSC, was found almost exclusively in
the late-proliferating subpopulation of
CD34+CD38 cells, providing further evidence
that the most primitive human hematopoietic cells are also the most
quiescent.
The switch culture system described in this report allows the assay of
cells with both lymphoid and myeloid potential, a characteristic considered a unique hallmark of HSC. The ability to expand HSC ex vivo
and to transfer therapeutic genes into HSC can now be studied directly
rather than relying on the surrogate assays of committed progenitors.
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FOOTNOTES |
Submitted October 20, 1997;
accepted February 3, 1998.
Supported in part by National Institutes of Health Grant Nos. HL54850
and CA-59318. G.M.C. is supported by a Translational Research Grant
from the Leukemia Society of America.
Address reprint requests to Gay M. Crooks, MD, Division of
Research Immunology/BMT, Childrens Hospital Los Angeles, MS #62, 4650 Sunset Blvd, Los Angeles, CA 90027.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We are grateful to the staff of the Labor and Delivery ward, Kaiser
Permanente Hospital Sunset (Los Angeles, CA) for their continuing essential contributions in the collection of cord blood samples; to Earl Leonard for biostatistical analysis; and to Drs Robertson Parkman and Donald B. Kohn for helpful advice on the manuscript.
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REFERENCES |
1.
Orlic D,
Bodine DM:
What defines a pluripotent hematopoietic stem cell (PHSC): Will the real PHSC please stand up!
Blood
84:3991,
1994[Free Full Text]
2.
Namikawa R,
Weilbaecher KN,
Kaneshima H,
Yee EJ,
McCune JM:
Long-term human hematopoiesis in the SCID-hu mouse.
J Exp Med
172:1055,
1990[Abstract/Free Full Text]
3.
Larochelle A,
Vormoor J,
Hanenberg H,
Wang JCY,
Bhatia M,
Lapidot T,
Moritz T,
Murdoch B,
Xiao XL,
Kato I,
Williams DA,
Dick JE:
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy.
Nat Med
2:1329,
1996[Medline]
[Order article via Infotrieve]
4.
Nolta JA,
Hanley MB,
Kohn DB:
Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: Analysis of gene transduction of long-lived progenitors.
Blood
83:3041,
1994[Abstract/Free Full Text]
5.
Nolta JA,
Dao MA,
Wells S,
Smogorzewska EM,
Kohn DB:
Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in imune deficient mice.
Proc Natl Acad Sci USA
93:2414,
1996[Abstract/Free Full Text]
6.
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 USA
94:5320,
1997[Abstract/Free Full Text]
7.
Gartner S,
Kaplan HS:
Long-term culture of human bone marrow cells.
Proc Natl Acad Sci USA
77:4756,
1980[Abstract/Free Full Text]
8.
Sutherland HJ,
Eaves CJ,
Eaves AC,
Dragowska W,
Lansdorp PM:
Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.
Blood
74:1563,
1989[Abstract/Free Full Text]
9.
Hao Q,
Shah AJ,
Thiemann FT,
Smogorzewska EM,
Crooks GM:
A functional comparison of CD34+CD38 cells in cord blood and bone marrow.
Blood
86:3745,
1995[Abstract/Free Full Text]
10.
Hao Q,
Thiemann FT,
Petersen D,
Smogorzewska EM,
Crooks GM:
Extended long term culture reveals a highly quiescent and primitive human hematopoietic progenitor population.
Blood
88:3306,
1996[Abstract/Free Full Text]
11.
Rawlings DJ,
Quan S,
Hao Q,
Thiemann FT,
Smogorzewska M,
Witte ON,
Crooks GM:
Production of human B-cell progenitors from highly purified cord blood CD34+, CD38 stem cells.
Exp Hematol
25:66,
1996
12.
Rawlings DJ,
Quan SG,
Kato RM,
Witte ON:
A long term culture system for selective growth of human B Cell progenitors.
Proc Natl Acad Sci USA
92:1570,
1995[Abstract/Free Full Text]
13.
Collins LS,
Dorshkind K:
A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis.
J Immunol
138:1082,
1987[Abstract/Free Full Text]
14.
Terstappen LWMM,
Huang S,
Safford M,
Lansdorp PM,
Loken MR:
Sequential generations of hematopoietic colonies derived from single nonlineage committed progenitor cells.
Blood
77:1218,
1991[Abstract/Free Full Text]
15.
Jordan CT,
Yamasaki G,
Minamoto D:
High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations.
Exp Hemat
24:1347,
1996[Medline]
[Order article via Infotrieve]
16.
Rusten LS,
Jacobsen SEW,
Kaalhus O,
Veiby OP,
Funderud S,
Smeland EB:
Functional differences between CD38 and DR subfractions of CD34+ bone marrow cells.
Blood
84:1473,
1994[Abstract/Free Full Text]
17.
Eaves CJ,
Sutherland HJ,
Cashman JD,
Otsuka T,
Lansdorp PM,
Humphries RK,
Eaves AC,
Hogge DE:
Regulation of primitive human hematopoietic cells in long-term marrow culture.
Semin Hemat
28:126,
1991
18.
Wineman JP,
Nishikawa S,
Müller-Sieburg CE:
Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: Effect of stromal cells and c-kit.
Blood
81:365,
1993[Abstract/Free Full Text]
19.
Wineman J,
Moore K,
Lemischka I,
Muller-Sieburg C:
Functional heterogeneity of the hematopoietic microenvironment: Rare stromal elements maintain long-term repopulating stem cells.
Blood
87:4082,
1996[Abstract/Free Full Text]
20.
DiGiusto DL,
Lee R,
Moon J,
Moss K,
O'Toole T,
Voytovich A,
Webster D,
Mule JJ:
Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo.
Blood
87:1261,
1996[Abstract/Free Full Text]
21.
Berardi AC,
Meffre E,
Pflumio F,
Katz A,
Vainchenker W,
Schiff C,
Coulombel L:
Individual CD34+CD38lowCD19CD10 progenitor cells from human cord blood generate B lymphocytes and granulocytes.
Blood
89:3554,
1997[Abstract/Free Full Text]
22.
Issaad C,
Croisille L,
Katz A,
Vainchenker W,
Coulombel L:
A murine stromal cell line allows the proliferation of very primitive human CD34+/CD38 progenitor cells in long-term cultures and semisolid assays.
Blood
81:2916,
1993[Abstract/Free Full Text]
23.
Hendry JH:
The f number of primary transplanted splenic colony-forming cells.
Cell Tissue Kinet
4:217,
1971[Medline]
[Order article via Infotrieve]
24.
Hodgson GS,
Bradley TR:
Properties of haematopoietic stem cells surviving 5-fluorouracil treatment: Evidence for a pre-CFU-S cell?
Nature
281:381,
1979[Medline]
[Order article via Infotrieve]
25.
Hodgson GS,
Bradley TR:
In vivo kinetic status of hematopoietic stem and progenitor cells as inferred from labeling with bromodeoxyuridine.
Exp Hematol
12:683,
1984[Medline]
[Order article via Infotrieve]
26.
Van Zant G:
Studies of hematopoietic stem cells spared by 5-fluorouracil.
J Exp Med
159:679,
1984[Abstract/Free Full Text]
27.
Rosendaal M,
Villa S,
Hooper C:
Correspondence between the development of hemopoietic tissue and the time of colony formation by colony-forming cells.
Blood Cells
12:615,
1987[Medline]
[Order article via Infotrieve]
28.
Ploemacher RE,
van der Slujs JP,
Voerman JSA,
Brons NHC:
An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse.
Blood
74:2755,
1989[Abstract/Free Full Text]
29.
Ploemacher RE,
van der Sluijs JP,
van Beurden CAJ,
Baert MRM,
Chan PL:
Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse.
Blood
78:2527,
1991[Abstract/Free Full Text]
30.
Traycoff CM,
Kosak ST,
Grigsby S,
Srour EF:
Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis.
Blood
85:2059,
1995[Abstract/Free Full Text]
31.
Berardi AC,
Wang A,
Levine JD,
Lopez P,
Scadden DT:
Functional isolation and characterization of human hematopoietic stem cells.
Science
267:104,
1995[Abstract/Free Full Text]
32.
Young JC,
Varma A,
DiGiusto D,
Backer MP:
Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture.
Blood
87:545,
1996[Abstract/Free Full Text]
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Abstract
Introduction
Abstract
