Characterization of Primitive Hematopoietic Cells in Normal Human
Peripheral Blood
By C. Udomsakdi, P.M. Lansdorp, D.E. Hogge, D.S. Reid, A.C. Eaves, and C.J. Eaves
The total number of clonogenic cells present in 5-week-old
long-term cultures (LTC) initiated by seeding normal human
marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic
input precursor cell type referred t o as an LTC-initiating cell
(LTC-IC). Previous studies have suggested that LTC-IC also
circulate because production of clonogenic cells continues
for many weeks when cells from the light-density ( ~ 1 . 0 7 7
g/mL), T-cell-depleted fraction of normal blood are maintained on irradiated, marrow-derived feeder layers in LTC
medium. We now show that the number of clonogenic cells
present in such reconstructed LTC after 5 weeks is linearly
related t o the input number of peripheral blood (PB) cells
over a wide range of cell concentrations, thereby permitting
the quantitation of circulating LTC-IC by limiting dilution
analysis. Using this approach, we have found the concentration of LTC-IC in the circulation of normal adults t o be 2.9 +:
0.5/mL. This is approximately 75-fold lower than the concentration of circulating clonogenic cells (ie, burst-forming unitserythroid plus colony-forming units [CFU] granulocytemacrophage plus CFU-granulocyte, erythroid, monocyte,
megakaryocyte) and represents a frequency of LTC-IC relative t o all nucleated cells that is approximately 100-fold
lower than that measured in normal marrow aspirate samples. Characterization studies showed most circulating LTC-IC
t o be small (low forward light scatter and side scatter),
CD34+, Rh-123du11,HLA-DR-, and 4-hydroperoxycyclophosphamide-resistant cells, with differentiative and proliferative potentialities indistinguishable from LTC-IC in normal
marrow. Isolation of the light-density, T-cell-depleted, CD34+,
and either HLA-DRIoWor Rh-123du1’fraction of normal blood
yielded a highly enriched population of cells that were 0.5%
t o 1% LTC-IC (-1,500-fold enriched beyond the light-density,
T-cell-depletion step), a purity comparable t o the most
enriched populations of human marrow LTC-IC reported t o
date. However, purification of PB LTC-IC on the basis of
these properties did not allow them to be physically separated from a substantial proportion (>30%) of the clonogenic
cells in the same samples, in contrast t o previous findings for
LTC-IC and clonogenic cells in marrow. These studies show
the presence in the blood of normal adults of a relatively
small but readily detectable population of functionally defined, primitive hematopoietic cells that share properties
with marrow LTC-IC, a cell type thought to have in vivo
reconstituting potential. They also serve t o emphasize the
point that phenotypic characteristics previously associated
with the most primitive hematopoietic cell types may not
necessarily correlate with their developmental potential,
underscoring the continuing importance of functional assays
in future studies of early events in hematopoiesis.
0 1992by The American Society of Hematology.
T
their ability to generate clonogenic progeny for periods in
excess of 5 weeks under the same culture conditions that
support CRU maintenance and proliferation.zo
The validity of the LTC-IC assay is dependent on the
existence of a linear relationship between the endpoint
measured (clonogenic cell output as assessed after 5 weeks
of culture) and the number of LTC-IC in the original test
suspension down to limiting numbers of LTC-IC. Previous
analyses of the cellular input required to achieve a sustained output of clonogenic cells for 5 weeks or more
indicated that, in the absence of added growth factors, two
cell types were required: the LTC-IC itself, and a second
ontologically unrelated “stromal” cell of the fibroblastendothelial-adipocyte lineage that provides an essential
H E PRESENCE OF primitive hematopoietic cells in
adult peripheral blood (PB) has been recognized for
three decades. Initial experiments showed that PB from a
variety of species, including humans, was capable of protecting recipients from lethal doses of whole body irradiation by
restoring blood cell formation from circulating donor
Subsequent studies led to the demonstration and quantitation of specific progenitor populations detected by colony
assays.6-s This prompted the evaluation of leukapheresis
harvests as an alternative source of cells for therapeutic
applications where autologous or allogeneic marrow transplants were not f e a ~ i b i e . ~More
- ’ ~ recently, identification of
strategies for increasing the concentration of clonogenic
progenitors in the circulation has heightened interest in the
potential of PB harvests for clinical treatment protocols
requiring hematologic rescue.13-16
Key to the future development of such protocols as well
as to an improved understanding of factors that regulate
stem cell mobilization, recruitment and marrow colonization is the availability of a quantitative assay for cells
capable of reconstituting and sustaining hematopoiesis in
transplant recipients. We have recently developed such an
assay for murine hematopoietic stem cells, based on the
demonstration of their competitive repopulating ability in
vivo after injection into recipients at limiting dilution.”
These multipotent competitive repopulating units (CRU)
are sustained in long-term cultures (LTC) of murine
marrow where at least some may undergo multiple selfrenewal divi~ions.’~J~
These findings strongly suggest overlap or even identity of CRU with cells referred to as
LTC-initiating cells (LTC-IC), the latter being defined by
Hood, Vol80, No 10 (November 15), 1992: pp 2513-2521
From the Teny Fox Laboratory, British Columbia Cancer Agency;
and the Departments of Pathology, Medicine, and Medical Genetics,
University of British Columbia, Vancouver, British Columbia, Canada.
Submitted April 16, 1992; accepted July 22, 1992.
Supported by a grant from the National Cancer Institute of Canada
(NCIC).C.U. was a Teny Fox Physician-ScientistFellow of the NCIC
and C.J.E. is a Teny Fox Research Scientist of the NCIC.
Address reprint requests to C.J. Eaves, PhD, Teny Fox Laboratory,
BC Cancer Research Centre, 601 W 10th Ave, Vancouver, BC,
Canada V5Z IL3.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I992 by The American Society of Hematology.
0006-4971I92 I8010-0009$3.00 I0
2513
2514
UDOMSAKDI ET AL
supportive f ~ n c t i o n . ~The
* - ~latter
~
proliferate in cultures
initiated with unseparated cell suspensions of normal bone
marrow (BM) and, if the initial number of BM cells present
is relatively high (ie, > lo6 cells/mL), will rapidly generate
sufficient progeny to support the LTC-IC coexisting in the
same original BM cell inoculum. However, to enable
detection of LTC-IC in suspensions that lack supportive
stromal cells or their precursors, as is the case for cells from
normal adult blood," a separate source of supportive cells
must be provided. We have previously shown that irradiated cells subcultured from the adherent layer of previously
established normal marrow LTC or from a competent
fibroblast cell line can be used for this p ~ r p o s e . ~ ~ , ~ ~
Under optimized assay conditions, LTC-IC in human
marrow can be quantitated by limiting dilution analysis21
and have been shown to have several characteristics of
quiescent cells. These characteristics include a relative
insensitivity to 4-hydroperoxycyclophosphamide (4-HC),25,26
low retention of rhodamine-123 (Rh-123),27small
and low expression or absence of CD71 (the transferrin
r e c e p t ~ r ) ~and
* , ~HLA-DR.20
~
At least a proportion of the
LTC-IC in normal adult human marrow show multipotentiality in the LTC system21and maintain their numbers at
levels comparable to CRU in analogous murine cult u r e ~ . ' ~Several
, ~ ~ years ago, we reported that myeloid
clonogenic cells were generated for at least 2 months in
LTC cultures initiated by seeding T-cell-depleted lightdensity PB cells onto irradiated, pre-established allogeneic
marrow-adherent layers,24suggesting the normal presence
of some LTC-IC in the circulation. In this study, we show
that these cells can be quantitated using the same conditions and limiting dilution procedure originally developed
and applied to the detection of LTC-IC in human marrow20J and that they also exhibit many of the same
properties, as expected of a very primitive, quiescent
hematopoietic cell.
MATERIALS AND METHODS
Cells. PB mononuclear cells were obtained with informed
consent from normal volunteer blood donors as a byproduct of
plateletphereses performed at the Vancouver General Hospital,
Canada. Cells were further depleted of T cells by incubation with
2-aminoethylbromide isothiouronium-treated sheep red blood cells
(RBCs) for 30 minutes at 4°C and subsequent isolation of the light
density ( < 1.077 g/mL) fraction after centrifugation on Ficollhypaque (FH) as described p r e v i o ~ s l yRandom
.~~
checking of this
procedure showed that less than 2% of the recovered cells were
CD2+ (T cells) by FACScan analysis. Normal BM aspirate cells
were obtained with informed consent from normal donors of
allogeneic marrow for transplantation. BM cells were either used
directly, or after lysis of contaminating RBCs by brief exposure to
ammonium
or after centrifugation on FH as indicated.
Cultures. Cells from primary blood or BM samples or from
LTC were assayed for erythroid (burst-forming units-erythroid
[BFU-E]), granulopoietic (colony-forming units-granulocytemacrophage [CFU-GM]), and multilineage (CFU-granulocyte,
erythroid, monocyte, megakaryocyte [CFU-GEMM]) colonyforming cells in standard methylcellulose cultures containing 3
U/mL of human erythropoietin and 10% agar-stimulated human
peripheral leukocyte-conditioned medium. This methodology and
the criteria used for colony recognition have been described in
detail previ0usly.3~LTC-IC assays were initiated by seeding an
aliquot of the test cell suspension into cultures containing irradiated (1,500 cGy) allogeneic marrow cells (3 x 1@/cm2) that had
been subcultured from the adherent layer of previously established
2- to 4-week-old LTC.20,34LTC-IC assay cultures were then fed
weekly by replacement of half of the growth medium containing
half of the nonadherent cells with fresh growth medium (a-medium
supplemented with inositol, folic acid, glutamine,
mol/L
2-mercaptoethanol,
mol/L hydrocortisone sodium hemisuccinate, 12.5% horse serum, and 12.5% fetal calf serum [FCS]). In
most experiments, LTC-IC assays were performed in cultures set
up in 2.5 mL volumes in 35-mm tissue culture dishes, although for
the limiting dilution assays, smaller, appropriately scaled down (0.1
mL) cultures were used as described previously.21After a total of 5
weeks (unless specified otherwise), the nonadherent cells were
removed, washed, and combined with cells harvested from the
adherent fraction by trypsini~ation.~~
These cells were then adjusted to a concentration suitable for plating in methylcellulose
assays (to yield <200 colonies per 1.1 mL assay culture). For a
detailed description of the LTC-IC assay procedure, see Eaves et
al.34In the experiments reported here (unless specified otherwise),
the number of clonogenic cells present in LTC harvested after 5
weeks (ie, the number of BFU-E plus CFU-GM plus CFU-GEMM
present in both the nonadherent and adherent fractions at this
time) was used to provide a quantitative, albeit relative, measure of
the number of LTC-IC originally seeded into the LTC. However, as
discussed in the Results, this number of clonogenic cells can be
directly converted to an absolute number of LTC-IC simply by
dividing by 4, because this is the average number of clonogenic cells
calculated to be present in 5-week-old cultures per initial LTC-IC
seeded.
Staining andflow cytometry. Cells were prepared for staining by
resuspension in Hanks' solution containing 2% FCS and 0.01%
sodium azide (HFN). They were then incubated with 1 to 2 &mL
of anti-HLA-DR-phycoerythrin (PE) (10' cells/mL) or Rh-123 at
a final concentration of 0.1 p,g/mL as described p r e v i o ~ s l yIn
.~~~~~
some cases, cells were stained with an anti-CD34 antibody (8G12)36
directly conjugated to PE or fluorescein isothiocyanate (FITC).
Stained cells were sorted using a Becton Dickinson FACStarPIUS
(fluorescence-activated cell sorter [FACS]) equipped with an
argon laser emitting at 488 nm (Becton Dickinson, Mountain View,
CA). Fluorescence of Rh-123-, FITC-, and PE-labeled cells was
measured using 530/30 and 575/26 band pass filters, respectively,
after calibration of the FACS before each sort using 10 Fm
fluorescent beads. In some experiments, cells were gated according
to their forward light scatter characteristics (FSC) and side scatter
characteristics (SSC) to exclude most erythrocytes and granulocytes, as described p r e v i o u ~ l y ?Cells
~ ~ ~ ~appearing in this light
scatter window (see Fig 4A, fractions I and 11) constituted greater
than 60% of the total light-density fraction of T-cell-depleted
blood cells. Cells were collected after sorting in Hanks' solution
containing 50% FCS and were maintained at 4°C until plated.
RESULTS
Quantitation of LTC-IC in normal blood. In a first series
of experiments, the number of clonogenic cells present
after 5 weeks in LTC initiated with T-cell-depleted suspensions of normal PB mononuclear cells seeded onto preestablished, irradiated marrow adherent layers was found
to be a linear function of the number of cells initially added
over a 1,000-fold range of input cell numbers. Results for a
representative experiment are shown in Fig 1. Three such
dose response experiments also included a series of assay
cultures (20 to 25 per point) that were seeded with limiting
2515
CHARACTERIZATION OF CIRCULATING STEM CELLS
Table 1. Quantitation of LTC-IC and Clonogenic Cells in Normal PB
Cell Type
BFU-E
CFU-GM
CFU-GEMM
LTC-IC
Concentration
(per mL)
170 t51 -c
4.6 t2.9 -c
20
5
0.6
0.5
Values for individual patients were calculated by multiplying the
progenitor frequency per lo5 cells by the total nucleated cell recovery
after both the T-cell-depletion and FH density centrifugation steps and
then again by the white blood cells per milliliter. Values shown are the
mean 2 SEM of data obtained from 23 different normal individuals.
I
*
.....a.
. ....... . ....... . ....... . .LLLUJ
I
lo4
I
I
I
105
io6
107
108
Initial Cells per LTC
Fig 1. Linear relationship between the number of light density
(<1.077g/mL) 1-celMepleted PB cells from a representativenormal
individual seeded onto pre-established, irradiated normal marrow
feeders and the total number of clonogenic cells detected when these
LTC were harvested and assayed in methylcellulose 5 weeks later.
The slope of the regressionline f i i e d to this data set is 0.92 f 0.09.
numbers of LTC-IC (ie, -1 LTC-IC per assay culture).
From the proportion of positive and negative assay cultures
(containing 2 1clonogeniccell each, or none, respectively),
absolute frequencies of LTC-IC in the original test cell suspension were calculated using Poisson s t a t i s t i ~ s(Fig
~ ~ ,2).
~~
1
cn
t3
a
U
.-a>,
1
Om3'
r
0
.-c0
5n
e
n
0.1
I
I
I
I
I
50
100
150
200
250
Initial cells per LTC (XIO")
Fig 2. Limiting dilution analysis of data from a representative
experiment in which decreasing numbers of light-density, T-celldepleted normal PB cells were seeded onto irradiated marrow feedem
and the number of clonogenic cells detectable after 5 weeks was then
determined. For this experiment, the frequency of LTC-IC in the
starting cell suspension (ie, the reciprocal of the concentration of test
cells that gave 37% negative cultures) was 1 per 1.5 x loscells (S%
confidence limits, 1 per 9.9 x l W to 1 per 2.2 x 106 cells).
From this value and a knowledge of the total number of
clonogenic cells produced by a large number of cells of the
same input suspension, the average 5-week output of
clonogenic cells per LTC-IC in normal blood was calcul.2,39 which is
lated. This value was found to be 3.7
similar to the value of 4.3 2 0.4 that we reported for
LTC-IC in normal BM.21Bulk measurements of the 5-week
clonogenic cell content of assay cultures initiated with
T-cell-depleted blood samples from other normal adults
could then also be used to derive absolute LTC-IC per
milliliter values using this average clonogenic output per
LTC-IC conversion factor. Table 1 shows the average
concentration of LTC-IC in the PB calculated from values
measured on 23 normal adults, together with the average
concentration of circulating clonogenic cells (BFU-E plus
CFU-GM plus CFU-GEMM) obtained for the same 23
samples. The derived value of approximately 3 LTC-IC/mL
is approximately 75-fold lower than the concentration of
circulating clonogenic cells both measured here (Table 1)
and reported p r e v i ~ u s l yHence,
. ~ ~ ~ ~the frequency of LTC-IC
relative to other nucleated cells in the blood (-1 per
2 x 106) is approximately 100-foldlower than the frequency
of LTC-IC relative to other cells in the BM.21
Phenotype of circulating LTC-IC. The distributions of
LTC-IC and clonogenic cells in various phenotypically
defined subpopulations of the T-cell-depleted, lightdensity fraction of normal PB were then assessed. These
were obtained using the FACS to separate cells on the basis
of their light scattering properties, expression of CD34,
HLA-DR, and Rh-123 uptake. As illustrated in Fig 3, even
after removal of the T cells from the light-density fraction
of leukapheresis samples, the frequency of cells expressing
readily detectable levels of CD34 (as defined by the vertical
gate shown in Fig 3B) was still very low (0.1% to 0.5%) as
compared with the non-T-cell-depleted light-density fraction of normal marrow, where values of 1% to 4% are
typically
even using similarly stringent gating
criteria?O Most of the cells in the fraction defined as CD34+
expressed no or low levels of HLA-DR (Fig 3C) and had
low SSC properties (Fig 3E and F). Cells with a CD34+ and
HLA-DRIOWphenotype (defined by the horizontal gate
shown in Fig 3C) were found almost exclusively among the
smallest light-density cells (low FSC, Fig 3E).
Figure 4 shows the distributions of LTC-IC and clonogenic cells observed when the total light-density T-cell-
*
UDOMSAKDI ET AL
2516
W
P
W
P
K
r
4I
4
Fig 3. Bhrariatecontour histograms of lightdensity, T-celldepleted normal PB cells stained
with anti-CD34 and anti-HLADR. (E) The distribution of these
cells in the low side scatter window (fraction I II in Fig 4A). (C)
The distribution of HLA-DRh@
and HLA-DR’OI tells after also
gating for CD34+ cells as indicated in (B). The light scattering
properties of CD34+HLA-DRtoW
and CD34*HlA-DRh* cells are
shown in (E) and (F), respectively. Unsorted, unstained control, and Irrelevant (1D3) antibody-stained cells are shown in
(0) and (A), respectively.
GREEN FLUORESCENCE
+
FORWARD LIGHT SCATIER
depleted fraction of PB cells was subdivided into three
populations defined by their light scattering properties:
I-low FSC, low SSC; 11-intermediate to high FSC, low
SSC; and 111-all remaining cells (ie, open FSC, intermediate SSC). Although each gated population contained approximately equal numbers of cells, virtually all LTC-IC and
most of the clonogenic cells were consistently found in the
fraction containing the smallest cells (I). No LTC-IC and
less than 5% of all clonogenic cells were found in fraction
111. Therefore, in subsequent sorts, only cells in the low SSC
fractions (I and 11) were analyzed.
k
A
Figure 5 shows the results of functional assays performed
on cells sorted both according to their expression of CD34
and HLA-DR. In this case, only CD34+cells were assayed.
These were then divided into HLA-DRh’phand HLA-DR””
subpopulations using the gates shown in Fig 3C. In some
experiments, CD34+HLA-DRIWcells were further subdivided into an HLA-DR- and an HLA-DRz population. No
LTC-IC and very few directly clonogenic cells were detected in the CD34+HLA-DRhighfraction. However, further subdivision of the remaining CD34+HLA-DRIWcells
did allow some differential separation of LTC-IC and
I
6
9 :
I
FORWARD LIGHT SCAlTER
I
11
m
Fig 4. (A) Ught scatter profiles of Tcell-deploted, Ilght-demrtynormal blood tells. (E) The mean 2 SEM of the percentages of nucleated t e l l s
(0).
clonogenic cells (0).
and LTC-IC (m) in each sorted fraction (n = 4).
CHARACTERIZATION OF CIRCULATING STEM CELLS
2517
100
T
A
B
-
80
60
40
20
0
HLA-DR PE
Fig 5. (A) A representative histogram of CD34'. light-density, T-celMepleted normal blood cells (inthe previously described low SSC window
shown in Fig 4A) double-stained with PE-conjugated antCHLA-DR. CD34*HLA-DRb cells were further subdivided into CD34'DR- (fraction 1) and
CD34*DR' (fraction 2) cells. The remaining cells are CD34*ML4-DIWh, indicated as fraction 3 in (A) and referred t o as CD34*DR+ cells in (B). The
clonogenic
,
cells (CI), and
dark histogram in (A) shows the profile for unstained cells. ( 6 )The mean f SEM of the percentages of nucleated cells (:I)
LTC-IC I.) in each sorted fraction (n = 3).
-
directly clonogcniccclls, morc of the lattcr ( -40% IJ 1 0 %
LTC-IC) being found in the HLA-DR' fraction. Tablc 2
shows thc cnrichmcnt and rccovcry valucs obtaincd for
LTC-IC and clonogcnic cclls in various HLA-DR subpopulations of the light scattcr-gatcd, CD34' fraction, as comparcd with thc unstaincd, light-dcnsity, T-ccll-dcplctcd
starting population in thcsc cxpcrimcnts. Rccovcry of
LTC-IC in thc CD34+HLA-DR1" fraction wasgrcatcr than
100% in all tivc cxpcrimcnts pcrformcd, suggcsting that all
circulating LTC-IC cxprcss rcadily dctcctablc lcvcls of
CD34. as do thosc in normal BM.'" The isolation of a rare
subpopulation of circulating cclls dcfincd by thc samc
propcrtics prcviously uscd to purify BM LTC-IC (ic,
low-density, low forward light scatter, high cxprcssion of
CD34, and low cxprcssion o f HLA-DR), allowcd a much
grcatcr cnrichmcnt ( > 1,000-fold bcyond thc light-density,
T-ccll-dcplction stcp) of circulating LTC-IC to bc routincly
obtaincd. Thus, cvcn though thc initial frcqucncyof LTC-IC
in normal PB is much lowcr (on a pcr ccll basis), the final
purity of LTC-IC achicvablc from normal PB using thcsc
Table 2. Frequency, Enrichment, and Recovery of LTC-IC and Clonogenlc Cells in Various Subpopulations of the CD34'. T-CeIl-Depletd,
Light-Density Fraction of Normal PB Cells Defined According t o Their Expression of MIA-DR
Cell Type
Evaluated
LTC-IC§
Frequency
Source
Unsorted cells"
DRhW
Clonogenic cells
DR'q
DR'"
DR-tt
Unsorted cells '
DRW9
DR1*
DR-**
DR-tt
1%).
0.0022 f 0.0004
O#
3.7 f 1.1
1.0 f 0.5
2.8 f 0.5
0.11 f 0.02
7.6 f 0.4
Enrichmentt
% Recovervt
-
-
1.930 2 470
540 t 270
1,470 z 340
300 t 40
48 t 36
280 f 17
-
65
5
22
NOS*
-
21 2 2
15 t 4
240 2 40
160 f 40
2.7 2 1.2
-
21
38
6
f5
2
No.of
Exoeriments
5
5
2
3
3
5
4
4
4
'Frequency of the cell type evaluated (LTC-ICor total clonogenic cells) relative to all nucleated cells in the population analyzed. (To convert the
LTC-IC frequencies shown to absolute frequencies, divide by 4).
tcalculated by dividing the progenitor frequency per 105 sorted cells by the progenitor frequency per lo5 unsorted, T cell-depleted, light-density
cells in each individual experiment, and then deriving the mean f SEM of these values for the number of experiments performed.
*Calculated by multiplying the percentage of cells retrieved in the fraction indicated by the corresponding calculated progenitor enrichment for
each individual experiment (defined in footnote c), and then deriving the mean 2 SEM of these values for the number of experiments performed.
§Measuredas the total number of clonogenic cells present after 5 weeks (ie, - 4 x the absolute LTC-IC number)
"Light-density, T-celldepleted cells.
AS defined in Fig 3C.
#None detected; ie, less than 0.15. Enrichment and recovery values are therefore not calculated.
"Refers to a subpopulation of CD34'HLA-OR'OI cells defined as fraction 2 in Fig 5A.
ttRefers to a subpopulation of CDM'HLA-DR'OI cells defined as fraction 1 in Fig 5A.
**Not done as a separate measurement. DR" recovery values can be inferred by adding together values for DR- and DR-.
2518
UDOMSAKDI ET AL
paramctcrs ( -0.5% to I%, Tahlc 2) was approximatcly thc
samc as thc bcst yct dcscribcd for normal RM.?1.27
Rccovcry
of clonogcnic cclls in thcsc samc cxpcrimcnts appcarcd to
bc somcwhat lowcr (Tahlc 2 ) , suggesting that somc circulating clonogcnic cclls may havc bccn cxcludcd from thc
CD34+ population gatcd for in thcsc studics, or that
suboptimal plating cficicncy of clonogcnic cclls may havc
heen achicvcd whcn highly purificd populations wcrc assaycd. Failurc to dctcct additional clonogcnic cclls in thc
highcr FSC/SSC fractions ( I 1 and Ill, Fig 4) duc to
potcntial inhibition of thcir colony-forming ability by thc
prcscncc of incrcascd numbers of monocytcs was rulcd out
by mixing cxpcrimcnts (ic, no rcduction of clonogcnic cclls
dctcctcd whcn cclls from fraction I wcrc mixcd with cclls
from fraction 111 in a 1:2 ratio: data not shown).
Thc rcsults of combincd staining for CD34 cxprcssion
and Rh-123 uptake arc shown in Fig 6. In this caw, no
diffcrcncc was notcd bctwccn circulating LTC-IC and
clonogcnic cclls in tcrms of thcir distribution bctwccn thc
CD34 Rh- 123du11and CD34+Rh- 123hrlpht fractions, with
morc than 80% of both bcing found in thc Rh-123du11
fraction. This contrasts with normal RM, in which most of
thc LTC-IC arc also Rh-123du11,
but most of thc clonhgcnic
cclls arc Rh-123*"pht, thus allowing for thcir diffcrcntial
isolation by sorting according to this paramctcr." NcvcrthcIcss, thc final purity of LTC-IC in thc light-dcnsity, T-cclldcplctcd, CD34+Rh-123du11
fraction of normal blood (data
not shown) was similar to that obtaincd by sclccting for
HLA-DRI" cclls (Tablc 2) or by application of thc samc
critcria to BM.?' This rcflccts a similarly grcatcr ovcrall
cnrichmcnt achicvcd with blood vcrsus marrow using cithcr
HLA-DR cxprcssion or rctcntion of Rh-123 as thc final
scparation paramctcr.
4-HC sensitivities of circulating progenitors. Bccausc normal circulating clonogcnic cclls wcrc known to bc a quicsccnt population" and appcarcd phcnotypically to bc morc
+
similar to LTC-IC in cithcr blood or BM than to thc
clonogcnic cclls found in thc RM. it was of intcrcst to
comparc thc scnsitivitics of circulating clonogcnic cclls and
LTC-IC to 4-HC, using thc samc typc of trcatmcnt protocol
that is in widcsprcad clinical usc for trcating autologous
marrow transplants. In this sct of cxpcrimcnts, LTC-IC
function (bcforc or aftcr cxposurc to 4-HC) was asscsscd in
tcrms of thc clonogenic ccll contcnt of assay culturcs
cvaluatcd aftcr 4 and 8 wccks (rathcr than aftcr 5 wccks as
in thc cxpcrimcnts dcscribcd abovc). bccausc prcvious
cxpcrimcnts had shown diffcrcnccs in LTC assays of BM
samplcs for autologous transplants whcn thcsc two timc
points wcrc comparcd.2s.?6Rcsults for LTC-IC and clonogcnic cclls in normal PB and BM arc shown in Fig 7. A
dramatic diffcrcncc in thc cffcct of 30 minutcs of cxposure
at 37°C to 100 wg/mL of 4-HC on thc viability of clonogcnic
cclls from blood and BM is apparent. Convcrscly, normal
circulating clonogcnic cclls and LTC-IC appear to bc
similar to BM LTC-IC in thcir rclativc rcsistancc to 4-HC.
For LTC-IC from both sourccs, a slight incrcasc in 4-HC
rcsistancc was notcd for LTC-IC dcfincd by thc longcr
clonogcnic ccll output endpoint (ic. 8 wccks).
Diflcrentiative potential cxpw.wed bv circitlating I, TC-IC in
LTC. Tablc 3 shows thc rclativc proportions of BFU-E,
CFU-GM. and CFU-GEMM in thc total clonogcnic population of 5-wcck-old LTC initiatcd with circulating LTC-IC
of varying puritics, and comparcs thcsc with thc rclativc
numbcrs of thcsc samc typcs of clonogcnic cclls in thc
original blood samplcs. Data for unscparatcd and LTC-ICcnrichcd ccll populations from normal BM obtaincd in
prcvious studics?"arc also shown in Tablc 3 for comparison.
I t can bc sccn that thc diffcrcntiativc bchavior cxhibitcd by
LTC-IC in normal blood and BM is similar and is also not
affcctcd by thc purity of thc LTC-IC in thc starting
population. In both cascs, a significant skcwing towards thc
gcncration of CFU-GM by comparison to thc numbcr of
Wl
A
40
20
RHODAMINE-123
CD34+Rh-l23dull CD34+Rh-123-bright
Fig 6. (A) A representativehistogram of CD34+, light-density, TtelMepleted normal blood cells double-stained with Rh-123 and sorted into
CD34*Rh-123M and CD34*Rh-123fractions (fractions 1 and 2, respectively). The dark histogramin (A) shows the profile for unstained cells.
(e)The mean i SEM of the percentages of nucleated cells ( Z ) ,clonogeniccells (0).and LTC-IC) .1 in each sorted fraction (n = 3).
2519
CHARACTERIZATION OF CIRCULATING STEM CELLS
1000 3
In
100 :
10
~
0
8
4
TIME IN CULTURE (WEEKS)
Fig 7. Comparison of the number of clonogenic cells and LTC-IC
surviving 30 minutes of exposure t o 100 pg/mL of 4-HC at 37°C with
7% erythrocytes present. Values shown are the mean f SEM of the
percentages of clonogenic cells and LTC-IC from normal BM (0)
and
normal blood (W) as a percent of values for control cells (n = 3 for BM
and n = 4 for blood cells).
CFU-GM and BFU-E actually found in normal blood or
BM was observed. To some extent this might be expected,
because all stages of granulopoietic cell differentiation are
supported in the LTC system, whereas erythropoiesis appears to be blocked at the stage of mature BFU-E product i ~ nAs
. ~a ~result, this latter contribution to total BFU-E
numbers in vivo is absent from LTC-derived populations.
DISCUSSION
We have previously described how the LTC system may
be used to quantitate and characterize a very primitive cell
in the BM of normal adults. Key to the interpretation of
data obtained with this approach is the use of a competent
feeder layer onto which the test cells are seeded so that the
endpoint of hematopoietic activity measured several weeks
later is determined solely and quantitatively by the number
of primitive hematopoietic cells (LTC-IC) initially present.
In addition, the duration of time allowed to elapse before
assessment of the hematopoietic cell content of the culture
and the level of differentiation of the hematopoietic cells
measured are important. A minimum of 5 weeks is required
for most input clonogenic cells to differentiate and disappear.20 Problems may also occur if quantitation of terminally differentiating granulocytes and macrophages are
used as a read-out of input hematopoietic potential because
the production of clonogenic cells and their subsequent
differentiation into mature progeny in these cultures appear to be differently regulated?* In the present study, we
have explored the usefulness of the LTC-IC assay originally
validated for human marrow cell suspensions for the
assessment of primitive hematopoietic cells in normal PB.
An additional requirement, noted previously, was the need
to rigorously remove T cells from the mononuclear fraction
of PB samples to be tested for their LTC-IC content to
circumvent the otherwise frequent spontaneous emergence
of rapidly growing Epstein-Barr virus (EBV)-transformed
lymphocytes during the 4- to 8-week period required to
complete the LTC-IC assay.”
Our results show that LTC-IC can be reproducibly
quantitated and found to be present, albeit at low levels, in
normal adult blood. Similar findings have also recently been
reported by Dooley and Law.44We have further shown that
normal circulating LTC-IC are indistinguishable from
LTC-IC in normal BM in terms of the number and types of
clonogenic progeny they produce and in terms of their
expression of CD34, ability to retain Rh-123, and sensitivity
to 4-HC. Two interesting differences, however, are the
apparent smaller size (lower FSC) and lack of detectable
expression of HLA-DR by most circulating LTC-IC. These
findings reinforce the concept that “stromal” cell-mediated
stimulation of long-term clonogenic cell production (> 5
weeks) in vitro allows the detection of a functionally distinct
primitive hematopoietic population that may, however, still
include some biologic and, hence, phenotypic heterogeneity. Interestingly, although the concentration of circulating
LTC-IC cells was found to be much lower ( 75-fold) than
the concentration of circulating clonogeniccells, the phenotypic characteristics of these functionally distinguished
populations were found to be very similar, as has been
previously shown for circulating clonogenic progenitors and
the subset that produces colonies of blast cells capable of
secondary colony formation in replating e~periments.4~9~
Whether circulating LTC-IC represent a related or even
-
Table 3. Relative Proportions of Different Types of Clonogenic Cells Detected Before and After 5 Weeks in LTC (% of total)
Original Progenitor
Clonogenic Cells‘
LTC-ICt
Source
No. of
Samples
BFU-E
CFU-GM
CFU-GEMM
BFU-E
CFU-GM
CFU-GEMM
Light-density fraction of normal blood*
LTC-IC-enriched fraction of normal blood§
Normal BMll
LTC-IC-enriched fraction of normal BMIJ
23
6
10
10
74 f 3
72 f 5
36 f 3
24 f 5
24 f 2
28 2 5
62 f 4
75 2 5
2.2 f 0.3
0.8 f 0.3
1.2 f 0.2
0.4 f 0.3
11 f 2
11 f 1
9 f2
9 f3
89 f 2
89 ~t2
91 f 2
90 f 4
0.5 f 0.2
1.02 0.6
0.8 f 0.3
1.4~
0.8
+Data shown are the mean f SEM of proportional values for specific clonogenic cell types expressed as a percent of all clonogenic cells (ie, BFU-E
plus CFU-GM plus CFU-GEMM) measured in standard short-term methylcellulose assays.
tData shown are the mean f SEM of proportional values for specific clonogenic cell types expressed as a percent of all clonogenic cells (ie, BFU-E
plus CFU-GM plus CFU-GEMM)measured in methylcellulose assays of cells harvestedfrom 5-week-old LTC.
*Same samples as in Table 1.
§Datafrom LTC-IC in fraction 1 (CD34+DR-)in Fig 5 (n = 3) and fraction 1 (CD34+Rh-123dUll)
in Fig 6 (n = 3).
IlData for normal BM from previously published studies.*O
2520
UDOMSAKDI ET AL
overlapping subset of progenitors detectable by any of the
previously described direct clonogenic cell assays remains
unresolved. Additional experiments t o look for the differential expression of other surface markers or growth factor
receptors on circulating LTC-IC and different types of
clonogenic cells may b e helpful in this regard.
It may also be possible eventually t o replace the adherent
fibroblast-like “stromal” cells in the LTC-IC assay by a
soluble source of t h e relevant factors they produce. Some
success along these lines has been reported.47 O u r own
more recent studies have shown that the self-maintenance
of LTC-IC can b e fully retained when LTC-IC are cultured
in the presence of Steel factor alone,48 although this can
also b e achieved when they are cocultured with murine
SI/SI fibroblast feeders that d o not contain the Steel gene.49
Such feeders, of course, also d o not produce any speciesspecific human factors (eg, granulocyte-macrophage colonystimulating factor [GM-CSF] and interleukin-3 [IL-3]),
suggesting that another, as yet undefined factor(s) can
support the maintenance a n d initial differentiation of
human LTC-IC. Clearly, further studies will be required to
establish the molecular identity of this factor(s), to determine whether a cellular mode of presentation is important,
and to evaluate whether LTC-IC in marrow and blood are
similarly regulated by such a factor(s).
Recently, we have shown that the LTC-IC assay can also
be used t o quantitate what appears t o be a developmentally
analogous primitive neoplastic (Ph*-positive) progenitor in
patients with chronic myeloid leukemia (CML).39 The
present studies thus also serve as an important baseline for
comparison with these CML LTC-IC and will likely facilitate future analyses of abnormal properties of neoplastic
stem cells in patients with other types of myeloproliferative
or myelodysplastic clones.
ACKNOWLEDGMENT
We thank Gayle Thornbury, Jessyca Maltman, and the support
staff of the Stem Cell Assay Service of the BC Cancer Agency and
the Cell Separator Unit at the Vancouver General Hospital for
expert technical assistance and aid in obtaining cell samples. We
also thank Don Henkelman for statistical advice and Judi Georgetti for typing the manuscript.
REFERENCES
1. Brecher G, Cronkite EP: Post-radiation parabiosis and survival in rats. Proc SOCExp Biol Med 77:292,1951
2. Goodman JW, Hodgson GS: Evidence for stem cells in the
peripheral blood of mice. Blood 19:702,1962
3. Epstein RB, Graham TC, Buckner CD: Allogeneic marrow
engraftment by cross circulation in lethally irradiated dogs. Blood
28:692, 1966
4. Storb R, Graham TC, Epstein RB, Sale GE, Thomas ED:
Demonstration of hemopoietic stem cells in the peripheral blood of
baboons by cross circulation. Blood 50:537, 1977
5. Abrams RA, Glaubiger D, Appelbaum FR, Deisseroth AB:
Result of attempted hematopoietic reconstitution using isologous,
peripheral blood mononuclear cells: A case report. Blood 56:516,
1980
6. McCredie KB, Hersh EM, Freireich EJ: Cells capable of
colony formation in the peripheral blood of man. Science 171:293,
1971
7. Chervenick PA, Boggs DR: In vitro growth of granulocytic
and mononuclear cell colonies from blood of normal individuals.
Blood 37:131,1971
8. Clarke BJ, Housman D: Characterization of an erythroid
precursor cell of high proliferative capacity in normal human
peripheral blood. Proc Natl Acad Sci USA 74:1105,1977
9. Korbling M, Dorken B, Ho AD, Pezzutto A, Hunstein W,
Fliedner TM: Autologous transplantation of blood-derived hemopoietic stem cells after myeloablative therapy in a patient with
Burkitt’s lymphoma. Blood 67529, 1986
10. Reiffers J, Bernard P, David B, Vezon G, Sarrat A, Marit G,
Moulinier J, Broustet A: Successful autologous transplantation
with peripheral blood hemopoietic cells in a patient with acute
leukemia. Exp Hematol 14:312, 1986
11. Juttner CA, To LB, Ho JQK, Bardy PG, Dyson PG, Haylock
DN, Kimber RJ: Early lympho-hemopoietic recovery after autografting using peripheral blood stem cells in acute nonlymphoblastic leukemia. Transplant Proc 2040,1988
12. Kessinger A, Armitage JO, Landmark JD, Smith DM,
Weisenburger DD: Autologous peripheral hematopoietic stem cell
transplantation restores hematopoietic function following marrow
ablative therapy. Blood 71:723,1988
13. Richman CM, Weiner RS, Yankee RA: Increase in circulating stem cells following chemotherapy in man. Blood 47:1031, 1977
14. Cline M, Golde W Mobilization of hematopoietic stem cells
(CFU-C) into the peripheral blood of man by endotoxin. Exp
Hematol5:186,1977
15. Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD: Granulocyte-macrophage colony stimulating
factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1:1194,1988
16. Gianni AM, Siena S, Bregni M, Tarella C, Stern AC, Pileri
A, Bonadonna G: Granulocyte-macrophage colony-stimulating
factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet 2580, 1989
17. Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC,
Eaves CJ: Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl
Acad Sci USA 87:8736,1990
18. Fraser CC, Eaves CJ, Szilvassy SJ, Humphries R K Expansion in vitro of retrovirally marked totipotent hematopoietic stem
cells. Blood 76:1071, 1990
19. Fraser CC, Szilvassy SJ, Eaves CJ, Humphries R K Proliferation of totipotent hematopoietic stem cells in vitro with retention
of long-term competitive in vivo reconstituting ability. Proc Natl
Acad Sci USA 89:1968,1992
20. 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
21. Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC,
Eaves CJ: Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive
marrow stromal layers. Proc Natl Acad Sci USA 87:3584, 1990
22. Sutherland HJ, Eaves CJ, Lansdorp PM, Thacker JD, Hogge
DE: Differential regulation of primitive human hematopoietic cells
in long-term cultures maintained on genetically engineered murine
stromal cells. Blood 78:666, 1991
23. Simmons PJ, Torok-Storb B: Identification of stromal cell
precursors in human bone marrow by a novel monoclonal antibody,
STRO-1. Blood 7855, 1991
CHARACTERIZATION OF CIRCULATING STEM CELLS
24. Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves
CJ: Unregulated proliferation of primitive chronic myeloid leukemia progenitors in the presence of normal marrow adherent cells.
Proc Natl Acad Sci USA 835306,1986
25. Winton EF, Colenda KW: Use of long-term human marrow
cultures to demonstrate progenitor cell precursors in marrow
treated with 4-hydroperoxycyclophosphamide.Exp Hematol 15:
710,1987
26. Eaves CJ, Sutherland HJ, Udomsakdi C, Lansdorp PM,
Szilvassy SJ, Fraser CC, Humphries RK, Barnett MJ, Phillips GL,
Eaves AC: The human hematopoietic stem cell in vitro and in vivo.
Blood Cells 18:301,1992
27. Udomsakdi C, Eaves CJ, Sutherland HJ, Lansdorp PM:
Separation of functionally distinct subpopulations of primitive
human hematopoietic cells using rhodamine-123. Exp Hematol
19:338, 1991
28. Sutherland HJ, Eaves CJ, Eaves AC, Lansdorp PM: Differential expression of antigens on cells that initiate haemopoiesis in
long-term human marrow culture, in Knapp W, Dorken B, Gilks
WR, Rieber EP, Schmidt RE, Stein H, von dem Borne AEG (eds):
Leucocyte Typing IV. White Cell Differentiation Antigens. Oxford, UK, Oxford, 1989, p 910
29. Lansdorp PM, Dragowska W: Long-term erythropoiesis
from constant numbers of CD34+ cells in serum-free cultures
initiated with highly purified progenitor cells from human bone
marrow. J Exp Med 1751501,1992
30. Eaves CJ,Cashman JD, Sutherland HJ, Otsuka T, Humphries
RK, Hogge DE, Lansdorp PM, Eaves AC: Molecular analysis of
primitive hematopoietic cell proliferation control mechanisms.
Ann NY Acad Sci 628:298,1991
31. Marsden M, Johnsen HE, Hansen PW, Christiansen JE:
Isolation of human T and B lymphocytes by E-rosette gradient
centrifugation: Characterization of the isolated subpopulations. J
Immunol Methods 33:323,1980
32. Turhan AG, Humphries RK, Phillips GL, Eaves AC, Eaves
CJ:Clonal hematopoiesis demonstrated by X-linked DNA polymorphisms after allogeneic bone marrow transplantation. N Engl J
Med 320:1655,1989
33. Cashman J, Eaves AC, Eaves CJ: Regulated proliferation of
primitive hematopoietic progenitor cells in long-term human
marrow cultures. Blood 66:1002, 1985
34. Eaves CJ, Cashman JD, Eaves AC: Methodology of longterm culture of human hemopoietic cells. J Tissue Culture Methods 1 3 3 , 1 9 9 1
35. Coulombel L, Eaves AC, Eaves CJ: Enzymatic treatment of
long-term human marrow cultures reveals the preferential location
of primitive hemopoietic progenitors in the adherent layer. Blood
62:291, 1983
36. Lansdorp PM, Sutherland HJ, Eaves CJ: Selective expression of CD45 isoforms on functional subpopulations of CD34+
hemopoietic cells from human bone marrow. J Exp Med 172:363,
1990
2521
37. Porter EH, Berry RJ: The efficient design of transplantable
tumour assays. Br J Cancer 17583,1963
38. Taswell C: Limiting dilution assays for the determination of
immunocompetent cell frequencies. I. Data analysis. J Immunol
126:1614,1981
39. Udomsakdi C, Eaves CJ, Swolin B, Reid DS, Barnett MJ,
Eaves AC: Rapid decline of chronic myeloid leukemic cells in
long-term culture due to a defect at the leukemic stem cell level.
Proc Natl Acad Sci USA 89:6192,1992
40. Sutherland HJ, Eaves AC, Eaves CJ: Quantitative assays for
human hemopoietic progenitor cells, in Gee AP (ed): Bone
Marrow Processing and Purging: A Practical Guide. Boca Raton,
FL, CRC, 1991, p 155
41. Ogawa M, Crush OC, O’Dell RF, Hara H, MacEachern
MD: Circulating erythropoietic precursors assessed in culture:
Characterization in normal men and patients with hemoglobinopathies. Blood 50:1081, 1977
42. Civin CI, Trischmann TM, Fackler MJ, Bernstein ID,
Buhring H-J, Campos L, Greaves MF, Kamoun M, Katz DR,
Lansdorp PM, Look AT, Seed B, Sutherland DR, Tindle RW,
Uchanska-Ziegler B: Report on the CD34 cluster workshop, in
Knapp W, Dorken B, Gilks WR, Rieber EP, Schmidt RE, Stein H
(eds): Leucocyte Typing IV. White Cell Differentiation Antigens.
Oxford, UK, Oxford, 1989, p 818
43. Eaves CJ,Eaves A C Cell culture studies in CML. Baillieres
Clin Haematol1:931,1987
44. Dooley DC, Law P: Detection and quantitation of long-term
culture-initiating cells in normal human peripheral blood. Exp
Hematol20:156,1992
45. Gabbianelli M, Sargiacomo M, Pelosi E, Testa U, Isacchi G,
Peschle C: “Pure” human hematopoietic progenitors: Permissive
action of basic fibroblast growth factor. Science 249:1561,1990
46. Gabbianelli M, Montesoro E, Pelosi E, Samoggia P, Sargiacomo M, Testa U, Valtieri M, Isacchi G, Peschle C: Pure
hemopoietic progenitors/stem cells from adult and cord blood:
Phenotype, growth factor (GF) requirement and modulation of the
expression of G F receptors (GFRs). Exp Hematol 19:465a, 1991
47. Brandt J, Srour EF, van Besien K, Briddell RA, Hoffman R:
Cytokine-dependent long-term culture of highly enriched precursors of hematopoietic progenitor cells from human bone marrow. J
Clin Invest 86:932,1990
48. Sutherland HJ, Reid D, Eaves CJ: Steel factor synergizes
with IL-3 and G-CSF to replace marrow feeders in promoting
differentiation of highly purified human hematopoietic cells. Exp
Hematol20755,1992
49. Sutherland HJ, Hogge DE, Cook DN, Eaves CJ: Long-term
maintenance of highly purified human stem cells on human and
murine fibroblast feeders is not influenced by conditions that either
increase or decrease exposure to steel factor. Blood 78:256a, 1991
(abstr, suppl 1)