1. No category

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Plastic-Adherent Progenitor Cells in Mobilized Peripheral
Blood Progenitor Cell Collections
By M.A. Scott, J.F. Apperley, H.K. Jestice, D.M. Bloxham, R.E. Marcus, and M.Y. Gordon
The useof peripheral blood progenitorcells (PBPC) t o reconstitute hematopoiesis after high-dosechemoradiotherapy is
now commonplace in the treatment of malignancies. Attempts t o characterize these cells have concentrated primarily on their phenotype and their content of clonogenic colony-forming cells (CFC).We have used a plastic-adherent
delta (PA) assay system t o evaluate the quantity and quality
of more primitivecells in addition to theconventional measurements ofCFC and CD34-positive cells. Theleukapheresis
products from 20 patients mobilized using cyclophosphamide (Cy) and granulocyte colony-stimulating factor (G-CSF)
were examined for progenitor cell content. The mean number of mononuclear cells (MNC), colony-forming units-granulocyte/macrophage (CFU-GM), and CD34-positive cells
from twoleukaphereses per patient were 7.9 x 108/kg, 47.3
x lO'/kg, and 10.5 x 106/kg, respectively. The mean number
of P A progenitors was 9.3 x 104/kg. Limiting dilutionanalyses showed thefrequency of P A progenitors in PBPC t o be
between 1 and 5.3 per I O 5 MNC and thateach P A progenitor
has the proliferative capability t o generate an overall mean
of 4.5 CFU-GM. Of the 20 patients, 16 underwent autografting with PBPC alone. Fifteen patients engrafted neutrophils
and platelets within 16 days. One patient had delayed engraftment associated with inadequate etoposide clearance.
Statistical analysis showed a strong correlation between
numbers of CFU-GM and CD34 positivity. The numbers of
plastic-adherent P A progenitor cells did not correlate with
CFU-GM or CD34-positive cells. We conclude that theplasticadherent P A progenitor cell assay is capable of measuring
primitive hematopoietic cells and that it may be useful for
the investigation of primitive progenitors in PBPC harvests.
0 7995 by The American Society of Hematology.
P
cell counts could be due tothe reinfusion o f a large spectrum
of differentiating progenitors in the leukapheresis product.
However, little is known of the characteristics and kinetics
of the cells with long-term repopulating ability that are harvested using this procedure. After cyclophosphamide treatment, with or without GM-CSF, the numbers of circulating
CD34-positivecellsandcolony-formingcells(CFC)
decrease toundetectable levels, and their reappearance together
with theappearance of long-termculture-initiating
cells
(LTC-IC) parallels white blood cell (WBC) recovery.13 I' We
have examined cellscollected by apheresis after mobilization
with cyclophosphamide and G-CSFto assess their progenitor
content using a recently devised assay for plastic-adherent
(PA) progenitor cells,Is in addition to the traditional colonyforming unit-granulocyte/macrophage (CFU-GM) assay and
quantitation of CD34-positive cells.
The fraction of bone marrow mononuclear cells that adheres to plastic contains progenitors capable of generating
substantialnumbers of CFU-GM in a l-week-longdelta
assay system. The plastic-adherent progenitors have, therefore, been called PA progenitors. Their early position in the
hematopoietic hierarchy has been confirmed by their ability
to generate long-term in vitro hematopoiesis, burst-forming
units-erythroid (BFU-E), andcobblestone area-forming cells
(CAFC)'" in addition toCFU-GM; theirresistance to 5fluorouracil''; their coexpression of the early phenotypic antigens CD34 and Thy- 1 "; and the absence of expression of
HLA-DR,CD33,andCD38.IX Removal of adherent cells
from murine marrow compromises its ability to reconstitute
irradiated recipients,"~" suggesting that adherence depletes
repopulatingcells. This studyhasassessedthefrequency
and proliferative potential of P A progenitors in mobilized
PBPC collections and compared them with CFU-GM and
CD34-positive cells as markers of progenitor cell content
and engraftment kinetics.
ERIPHERAL BLOOD progenitor cells (PBPC) mobilized by chemotherapy, with or without hematopoietic
growth factors (HGFs), are beingused increasingly to reconstitute hematopoiesis after high-dose chemoradiotherapy for
avarietyofhematologic
and other malignancies."' It has
beenknown for some time that primitive progenitor cells
circulate in low numbers in steady-state peripheral
and their numbers increase dramatically after various treatm e n k c In mice, the numbers of repopulating stem cells have
been shown to increase during the recovery phase after chemotherapy,' and after the administration of an HGF such as
granulocyte colony-stimulating factor (G-CSF). In humans,
G-CSF"'" and granulocyte-macrophage (GM)-CSF" are the
most widely used cytokines in mobilization protocols. The
major advantage of PBPC transplantation over conventional
autologousbone marrowtransplantation
(ABMT) isthe
more rapid recoveryof peripheral blood neutrophil and plateof pancytopenia
let counts, therebyshorteningtheperiod
thatis responsibleforthe
life-threateninginfections and
hemorrhage associated with transplantation. A reduction in
a
therequirement for antibiotics and bloodproductsand
shorterhospitalstay
can result in an overall costsaving
as compared with ABMT," although the financial benefits
remain somewhat controversial.
It is believed that the rapid recovery of peripheral blood
From the Department of Haematology, Addenbrooke's NHS Trust,
Cambridge; and the Department of Haematology, Royal Postgraduate Medical School, Lnndon, UK.
Submitted November 29, 1994; accepted July 31, 1995.
Supported by The Leukaemia Research Fund of Great Britain and
The Kay Kendall Leukaemia Fund.
Address reprint requests to M.A. Scott, Bone Marrow Transplant
Unit, Department of Haematology, Addenbrooke's NHS Trust, Hills
Road, Cambridge CB2 2QQ. UK.
The publication costs of this article were defrayedin part by page
chargepayment.Thisarticle
must thereforebeherebymarked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8612-0023$3.00/0
4468
MATERIALS AND METHODS
Patients and PBPC collection. Twenty consecutive patients underwent PBPC collection and subsequent autografting
in Cambridge,
UK, between April and August
1994. All gave informed consent.
Diagnoses included non-Hodgkin's lymphoma (NHL; I I patients),
Blood, Vol 86, No 12 (December 15). 1995: pp 4468-4473
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CELLS
PLASTIC-ADHERENT
IN PBPC COLLECTIONS
4469
Table 1. Patient Details and Progenitor Cell Yields
Engraftment
No. of CelldProgenitors Harvested
UPN
21 1
225
230
235
240
244
2 46
~i~~~~~
MM
MM
MM
MM
MM
”
AB
MM
MM
207
213
219
221
222
223
226
228
242
243
245
239
NHL
NHL
NHL
NHL
NHL
NHL
NHL
NHL
NHL
NHL
NHL
HD
MNC X 108/kg
6.3
13.2
16.4
17.7
3.0
16.0
8.5
8.1
5.8
4.2
9.3
4.2
7.3
5.6
5.6
5.3
9.0
5.9
6.9
6.5
CFU-GM
X
10‘/kg
48.0
95.5
140.5
31.5
17.1
193.6
1.8
40.2
13.7
5.1
31.1
47.9
18.7
24.6
11.3
12.0
74.1
12.2
38.2
16.2
CD34+ Cells x 1O6/kg
n/a
24.9
13.1
14.2
3.8
44.6
NA
10.2
NA
NA
12.6
6.4
2.8
2.1
3.2
4.8
9.8
2.9
5.7
5.5
PA Cells x 104kg
Count
10.4
2.7
36.8
7.3
12.6
23.5
0.5
9.0
28.6
0.9
5.0
5.8
3.9
6.1
3.5
4.9
5.8
7.3
7.7
4.1
Days t o Neutrophil
> 0.5 x 1 o V ~
Days t o Platelet
Count > 50 x 10%
12
12
25
10
9
45
-
-
11
10
12
9
-
-
-
-
10
11
-
-
12
11
13
14
13
11
12
11
14
14
10
16
13
13
14
15
16
14
13
12
Reinfusion doses given as per kilogram body weight. Engraftment data were excluded from patients 213 and 246 who received autologous
bone marrow in addition to PBPC, patient 235 who received CD34-purifiedcells, and patient AB who did not undergo autografting and was not
assigned a UPN. There were no statistical differences between the numbers of progenitor cells collected from the patients with myeloma and
the patients with lymphoma (CFU-GM,P = .49; CD34, P = .49; PA, P = .47).
Abbreviations: MM, multiple myeloma; NA, results not available.
Hodgkin’s disease (HD; one patient), and multiple myeloma (eight
patients). Patient characteristics are listed in Table 1. The patients
with lymphoma were a heavily pretreated cohort, while themyeloma
group had received induction chemotherapy with vincristine, Adriamycin, and dexamethasone (VAD) but no other chemotherapy. All
patients were primed with cyclophosphamide (Cy; Farmitalia Carlo
Erba Ltd. St Albans, UK) 3 g/m2 and G-CSF (Amgen Ltd, Cambridge, UK) 5 pg/kg/d to mobilize their PBPC. Cells were harvested
on 2 consecutive days when the recovering WBC count surpassed
4 X 1Oy/L. Usinga Cobe Spectra continuous flow apheresis machine,
three times the patient’s estimated blood volume was processed via
a double lumen central line of dialysis quality (Quinton Mahurker,
Seattle, WA) using the manufacturer’s WBC set (Cobe 777 006
000). The procedure took between 3 and 4 hours and used ananticoagulant-to-blood ratio of 1: 14. Flow rates varied between 60 and 80
mL/min, and a separation factor of 900 was used. Throughout the
collection, the hematocrit of the harvested cells was maintained between 2%and 4%, and the resultant mononuclear cell volume ranged
from 140 to 180 mL. Red cell contamination was less than 5% in
all cases. The cells were frozen in a rate-controlled freezer and stored
in the vapor phase of liquid nitrogen. The cryopreserved-thawed cells
were reinfused after conditioning therapy using the BEM regimen.”
Briefly, this consists of BCNU 300 or 450 mg/m2 (according to lung
function tests), etoposide 2 g/m’. and melphalan 140 mg/m’.No
hematopoietic growth factors were administered after the autograft.
Venous blood was taken from 10 laboratory volunteers as controls.
CFU-CM assay. Mononuclear cells (MNC) from each leukapheresis were isolated by density-gradient separation over Histopaque-1.077 (Sigma Diagnostics, Poole, UK). The cells from the
interface were harvested and washed twice in Hank’s buffered salt
solution (HBSS; GIBCO, Paisley, UK). They were resuspended in
1 mLof HBSSand counted. Fresh viable MNC were plated in
duplicate in 35-mm Petri dishes (Nunc, Life Technologies, Ltd, Paisley, UK)in semisolid medium containing 1.0% methylcellulose
(Fluka, Buchs, Switzerland) in Iscove’s modified Dulbecco’s medium (GIBCO) 30% fetal calf serum (FCS; GIBCO), 1 % bovine
serum albumin (Sigma Diagnostics), P-mercaptoethanol ( 5 X
m o m ; Sigma Diagnostics), and recombinant GM-CSF (50 IU/mL;
Schering-Plough, Mildenhall, UK) asa source of hematopoietic
growth factor. The MNC were incubated at 37°C in humidified 5%
CO, in air for 14 days. Colonies of more than 50 cells were scored
on day 14. Results were expressed as the number of CFU-GM per
10’ MNC plated and converted to a reinfusion dose per kilogram of
body weight.
Deltaassay of plastic-adherent (PA) progenitorcells.
MNC
from the PBPC collections were isolated by density-gradient separation. Ten million MNC were suspended
in 10 mL a-medium (GIBCO)
supplemented with 15% fetal calf serum and incubated at 37°C in
humidified 5% CO2 in air for 2 hours in 25-cm plastic tissue culture
flasks (Nunc). Preliminary experiments showed that increasing the
incubation period to 18hours did not increase the number of progenitor cells that adhered to the flask (unpublished data). Nonadherent
cells were removed by washing the flasks three times with HBSS.
Next, a-medium supplemented with 10% FCS, 10%horseserum
(GIBCO), 10% 5637 conditionedmedium,”and
2 X IO-‘ m o m
methylprednisolone (Upjohn, Crawley, UK) was added to the flasks
containing the residual adherent cells. The flasks were incubated at
37°C in humidified 5% CO2 in air for 7 days. The nonadherent cells
were harvested and assayed for CFU-GM. Colonies of more than
50 cells were scored on day 7; of more than 200 cells, on day 14;
and of more than 500 cells, on day 21.” Colonies were expressed
as the number of CFU-GM per flask per lo7 cells plated and backcalculated to give the number of PA progenitor cells present per
milliliter of blood; and hence, the numbers reinfused.
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4470
SCOTT ET AL
200
-
175
-
150
-
Limiting dilution analysis. Limiting dilution analysis wasused
to establish the frequency and proliferative potential of the PA progenitors in the PBPC collections. The assays were performed in 96well plates (Nunc) using 100-pL volumes in a scaled down version
of the bulk cultures in flasks. In five experiments, five mononuclear
cell concentrations (lo5,3 X IO4, IO4, 3 X IO3, and IO’) were added
to at least 32 replicate wells per group. The frequency of the PA
progenitors was calculated according to TaswelL2’ The number of
CFU-GM produced per individual PA progenitor cell was evaluated
from wells with a probability of monoclonality of more than 75%.
In one experiment, 96 replicate wells wereusedto
confirm the
distribution obtained from the other experiments.
CD34-positive enumeration. Unpurified PBPC were incubated
for 15 minutes with directly conjugated monoclonal antibodies
CD45-fluorescein isothiocyanate (FITC; HLe-l clone; Becton Dickinson, Cowley, UK) and CD34-phycoerythrin (PE; HPCA-2 clone;
Becton Dickinson). Red cells were lysed using Facslyse (Becton
Dickinson). Twenty thousand CD45-positive events were analyzed
for CD34-PE positivity using a Coulter EPICS Elite flow cytometer
(Coulter Electronics, Luton, UK). Only those cells clustering with
low side scatter properties were included in the analysis. The results
were expressed as the percentage of the total leukocytes collected
and converted to the absolute number of CD34-positive cells per
collection.
Statistical analysis. All statistical analyses were performed using a computer statistical package (Statview; Abacus Software,
Berkeley, CA). Simple linear regression analysis was used to examine potential correlations. Statistical significance was assessed using
the Mann Whitney U test, Wilcoxon signed rank test, and the
Kruskal-Wallis test for multiple samples.
respectively. The numbers of MNC and progenitors harvested are listed in Table 1. There was a significant correlation(Fig 1) between the numbers of CFU-GM and
CD34-positive cells ( r = 36: P = .001), but thenumbers
of plastic-adherent PA progenitor cells did not correlate with
CFU-GM or CD34-positive cells.
Quuntitution of PA progenitors. We first examined PA
cultures from the peripheral blood of 10 normal volunteers.
Four of the I O volunteers produced less than one CFU-GM
per flask per 10’ mononuclear cells plated. The remainder
producedbetween 10 and SO CFU-GM. A single limiting
dilution experiment on normal blood gave a frequency of l /
10” mononuclear cells. Four limiting dilution analyses of
PBPC gave frequencies of PA progenitor cells of between
I 2 0.3 (mean i- SE: 95% range, 0.3 to 1.6) and 5.3 t 0.7
(3.8 to 6.7) per IO’ MNC, which is similar to that of normal
bone marrow (2.5 to 10 per IO’ MNC).” The fifth analysis
gave a frequency of 0.3/10h, whichis similar tonormal
blood, and because only 3%of thewellswere
positive,
this analysis was excluded from further calculations. The
proliferative potential of individual PA progenitors was heterogeneous with respect to CFU-GM production within experiments, but the distributions for different samples were
very similar ( P = 3 , Kruskal-Wallis Test; Fig 2). Consequently, it seemed justified to calculate the number of PA
progenitor cells present in the bulk cultures by dividing the
total number of CFU-GM produced by the mean of the individual values (2.4, 3.4, 5.1, and 7.1) for CFU-GM per PA
progenitor cell (ie, 4.5 i 1.8). The derived number of PA
cells infused is listed in Table 1.
Engraftment kinetics. Of the 20 patients who underwent
PBPC mobilization and collection, four were excluded from
further evaluation. The cells from one patient (unique patient
number [UPN] 235) were further manipulated to obtain purified CD34-positive cells, two patients (UPN 213 and 246)
yielded low numbersof CFU-GM and subsequently received
stored back-up autologous bone marrow together with their
PBPC, and one patient (AB) relapsed and did not undergo
autografting.
All patients who received PBPC alone were given more
than our threshold number of 10 X l O4 CFU-GM24and more
than 2.7 X 1O4 PA cells per kilogram. Fifteen of 16 patients
engrafted rapidly, with a median of 12 days to a neutrophil
count of greater than 0.5 X I O ’ L and 12 days to a sustained
platelet count of greater than 50 X 10’L. The remaining
patient (UPN 230)had delayed engraftment (25 and 45
days), whichwas associated withpoorrenal clearance of
etoposide. Infusion of more than the threshold number of
cells and consistent rapid engraftment precluded evaluation
of an association between progenitor cell numbers and engraftment kinetics.
RESULTS
DISCUSSION
0
M
3
125-
%i
100-
2
75-
2
5:
[?
V
50
-
CD34-positive cellsx lo6per kg
Fig 1. Correlation between the numbers of CFU-GM and CD34positive cells in the leukapheresis harvests (n = 16).
PBPC harvesting. The median time taken for the recovering WBC count to reach 4 X 109Lwas 10 days (range, 9
to 13 days). The mean number of MNC collected from two
leukaphereses was 7.9 X 108/kg, of which 10.5 X 106/kg
were CD34-positive. Mean numbers of day-l4 CFU-GM
and PA progenitors were 47.3 X 104/kg and 9.3 X 104/kg,
Small numbers of primitive hematopoietic progenitor cells
are known to circulate in steady-state peripheral b l ~ o d .In
~.~
the recovery phase after chemotherapy or after administration of certain HGFs, numbers increase to levels that allow
collection and subsequent autografting. For reasons that are
not entirely clear, engraftment after PBPC transplantation is
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441 1
PLASTIC-ADHERENTCELLS IN PBPCCOLLECTIONS
Day 14
Day 7
100 l
"
19
0
5
U
Fig 2. Cumulative distribution of CFU-GM numbers (days
7,14, and 21 and total) produced
by individual P A progenitors
cultured at limitingdilution. The
data plotted were taken from
the cell concentration that corresponded to a probability of
greater than .75 that the contents of the wells were monoclonal. Theresults are from three
separate experiments.
They axis
shows the percentage of positive wells yielding between 1
and n CFU-GM; results from the
negative wells are not plotted.
The individual curves represent
results from 7 to 12 wells. The
frequenciesofnegative
wells
were 0.63, 0.75, and 0.78; the
probabilities of monoclonali
were SO, .75, and .78. In one experiment (data not plotted)consisting of 96 replicate wells,
11.5% of the wells were positive
(probability of monoclonality =
.94) and produced a total of 1 to
11 CFU-GM.
10
15
0
20
5
10
15
20
n CFW-GMper well
CFUGM pet well
Day 2I
Total
2B
2e,
*B
0
5
10
15
n Cm-GM per well
more rapid than with bone marrow, presumably as a result
of infusing a spectrum of differentiating cells. This procedure
has radically altered the clinical approach to high-dose therapy and stem cell rescue. The kinetics and characteristics of
PBPC are under intensive investigation and are as yet not
fully understood. Much of this work has focused on CD34positive cells and CFU-GM, but the long-term culture system, which requires bone marrow stroma as a feeder layer,
has been used to study more primitive progenitor cells.I4 A
recently described assay takes advantage of the adherent
nature of primitive hematopoietic cells in bone
When cultured in a plastic tissue culture flask, the nonadherent component that contains CFU-GM is removed, leaving
earlier progenitors adherent to the side of the flask. Subsequent CFU-GM production can, therefore, be attributed to
the pre-CFU-GM that adhered to the flask. It is possible to
20
n CFU-GMper well
measure C m - G M after 7 days because preexisting CFUGM are removed at the outset and because stromal cells,
which inhibit CFU-GM prod~ction?~
are not present to any
significant degree in the first 7 days of culture. We have
used this assay to measure the frequency and proliferative
capacity of these cells in PBPC collections.
Limiting dilution analysis has shown that the frequency
of PA progenitor cells in PBPC collections is similar to that
of normalbonemarrow (ie, 1 to 5.3 v 2.5 to 10 per IO5
MNC, re~pectively),'~
but their capacity to generate CFUGM is markedly less. Comparison of the distribution of the
numbers of CFU-GM produced by PA cells in PBPC with
the distribution of the numbers produced by PA cells in
normal bone marrowL5reveals a significant difference (P =
.001, Wilcoxon signed rank test). The overall mean value of
4.5 2 1.8 CFU-GM per PA progenitor cell that we have
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4472
measured for PBPC can be compared with a mean value for
normal marrow (n = 9) of 24 t 14 (Gordon" and unpublished data, 1995; P = ,005, Mann-Whitney U test). This
could reflect thepresence of moremature PA progenitor
cells in the PBPC harvests compared with bonemarrow,
which may only be capable of a small number of divisions
before terminaldifferentiation.Alternatively,the
PA cells
from PBPCmay undergo undetectable self-renewaldivisions
resulting in lower outputof CFU-GM in the short timeperiod
used for the assay. Insupport of this suggestion, the cumulative distribution of CFU-GM production (Fig 2) is similar
to that for PA cells in human umbilical cord blood (HUCB),
whichhavebeenshown
tobecapable
of self-renewal.'"
Sutherland et all4 have reported a similar phenomenon with
regard to LTC-IC in PBPC collections, which produce fewer
CFU-GM than LTC-IC in normal bone marrow.
The heterogeneity of CFU-GM production by individual
progenitor cells is a common feature of several hematopoietic cell assay system~.''.~'.~'Oneexplanation isthatthe
heterogeneous progenitor cell population represents a spectrum of cells with different degrees of maturity and ability
to generate more mature progeny. Another is that cells undergo self-renewal or differentiation according to a stochastic distribution of probability. It is tempting to speculatethat
the probability of P A progenitor cell self-renewal in PBPC
and HUCB is greater thanthat of P A cells in adult bone
marrow. This would imply that PBPC collected after mobilization therapy contain a population of cells that are more
capable of amplification than those found in a bone marrow
harvest. It is relevant in this context that PBPC have been
identifiedasidealtargets
for retroviralgene transfer." It
may be thattheseprimitiveprogenitor
cells are normally
held in microenvironmental niches and are not released during bone marrow sampling.However,chemotherapyand
growth factor administration may alter expression of adhesion proteins and result in the detachmentof these cells from
the stromal compartment.
Although CFU-GM production by individual P A cells in
PBPC is heterogeneous, the range of clonogenic production
is fairly narrow withineach experiment, and the
distributions
obtained from theindividual experiments were indistinguishable statistically. It was, therefore, reasonableto use the
overall mean of 4.5 CFU-GM per individual precursor cell,
obtained from four limiting dilution
experiments, to estimate
the number of PA progenitor cells inbulkcultures.
The
number of CFU-GM per PA cell is slightly higher than the
I .6 seen with LTC-IC."
Of the 16 patients whounderwent autografting with PBPC
alone, 15 engraftedneutrophils and plateletsrapidlywith
no subsequent transient decrease in counts. There was no
statistical difference between engraftment times and
parameters associated with progenitor cells. Only one patient (UPN
230) haddelayed engraftmentdespite receiving adequate
numbers of CFU-GM (140.5 X 104/kg). This was believed
to be due to inadequate clearanceof etoposide as a result of
deranged renal function associated with renal amyloidosis.
We found no correlation between numbers
of primitive
P A cells in our assay system and the number of CFU-GM
orCD34-positivecells presentin the harvests.Similarly,
SCOTT ET AL
Sutherland et all' did not find a correlation between LTCIC and CFU-GM or CD34-positive cells. This is not unexpected, as these two primitive populations are much smaller
fractions of the total CD34-positive population than are
CFU-GM. Results from individual patients in thestudy
showed that PA progenitors are present in PBPC collections
to varying degrees. In the twopatients who had low numbers
of CFU-GM (our target value is 10 X I O'/kg), PA cells were
0.5 X IO'//lig and 0.9 X 1O4/kg.The lowest number of PA
cells measured in a patient who engrafted successfully was
2.7 X lO''/kg. It may be that the minimum required number
lies between 0.9 X IO'Ikg and 2.7 X IOJ/kg. However, this
is difficult to demonstrate,as the majority of patients recover
their counts within 16 days. Although there is no correlation
evident between P A cells and engraftment, or
between LTCIC andengraftment, there may be a thresholdeffect for
primitive cells similar to that for the numbers of CFU-GM
infused.*4
Weconclude thattheplastic-adherentprogenitor
cell
assay is capable of measuring the quality and quantity of
primitive cells that are ancestral to CFU-GM in the hematopoietic hierarchy.
ACKNOWLEDGMENT
We are grateful to Dr N.M. Blackett for assistance with statistics.
REFERENCES
1.BriceP,Marolleau
JP, Dombret H, Lepage E, Baruchel A.
Adam M, Miclea JM, Sitthy X, Gisselbrecht C: Autologous peripheral blood stem cell transplantation after high dose therapy
in patients
with advanced lymphomas. Bone Marrow Transplant 9:337, 1992
2. Reiffers J, Marit G, Boiron JM, Rice A, Bernard P, Vezon G:
The role of peripheral blood stem cells as rescue after myeloablative
therapy. Bone Marrow Transplant 1:212. 1989
JM, DeChristopherPJ.
3.Williams SF, BitranJD,Richards
BarkerE,Conant J, GolombHM,Orlina AR: Peripheral bloodderived stem cell collections for use in autologous transplantation
after high dose chemotherapy: An alternative approach. Bone Marrow Transplant 5: 129, 1990
4. GoodmanJW,Hodgson CS: Evidence for stem cells in the
peripheral blood of mice. Blood 19:702, 1962
5. McCredie KB, Hersh EM, Freireich EJ:Cells capableof colony
formation in the peripheral blood of man. Science 171:293. 197 I
6 . Gordon MY: Physiological mechanisms in BMTand haemopoiesis-revisited.BoneMarrowTransplant 11:193, 1993
7. Craddock CF, ApperleyJF,WrightEG,HealyLE,
Bennett
CA, Evans M, Grimsley PG, Gordon MY: Circulating stem cells i n
mice treated with cyclophosphamide. Blood 80:264, 1992
8. Duhrsen U, Villeval JL, Boyd J, Kannourakis G, Morstyn G ,
Metcalf D: Effect of recombinant human granulocyte colony-stimulating factor onhematopoieticprogenitor cells in cancer patients.
Blood 72:2074, 1988
9. Lindemann A, Herrmann F, Oster W: Hematologic effects of
recombinant human granulocyte colony-stimulating factor
in patients
with malignancy. Blood 74:2644, 1989
10. MatsunagaT,Sakamaki S, Kohgo Y , Ohi S, Hirayama Y ,
Niitsu Y: Recombinant human granulocyte colony-stimulating factor
can mobilize sufficient amounts of peripheralbloodstem cells in
healthy volunteers for allogeneictransplantation. Bone Marrow
Transplant 1 1:103, I993
I 1. Gianni AM, Siena S, Bregni M, Tarella C, Stem AC. Pileri A,
Bonadonna G: Granulocyte-macrophage colony-stimulating factorto
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
PLASTIC-ADHERENT CELLS IN PBPC COLLECTIONS
harvest circulating haemopoietic stem cells for autotransplantation.
Lancet 2581, 1989
12. Ager S, Scott MA, Mahendra P, Richards EM, Jestice HK,
Boraks P, Baglin TP, Marcus RE: Peripheral blood stem cell transplantation after high-dose therapy in patients with malignant
lymphoma: A retrospective comparison with autologous bone marrow transplantation. Bone Marrow Transplant 16:79, 1995
13. Pettengell R, Testa NG, Swindell R, Crowther D, Dexter
TM: Transplantation potential of hemopoietic cells released into the
circulation during routine chemotherapy for non-Hodgkins
lymphoma. Blood 82:2239, 1993
14. Sutherland HJ, Eaves CJ, Lansdorp PM, Hogge DE: Kinetics
of committed and primitive blood progenitor mobilization after chemotherapy and growth factor treatment and their use in autotransplants. Blood 83:3808, 1994
15. Gordon MY: Plastic-adherent cells in human bone marrow
generate long-term hematopoiesis in vitro. Leukemia 8:865, 1994
16. Gordon MY, Lewis JL, Blackett NM, Amos TAS: Adhesive
phenotypes of primitive human haemopoietic progenitor cells. Br J
Haematol 87:96, 1994 (abstr)
17. Gordon MY, Riley GP, Greaves MF: Plastic-adherent progenitor cells in human bone marrow. Exp Hematol 15:772, 1987
18. Grand FH, Marley SB, Lewis JL, Goldman JM, Melo JV,
Gordon MY: Phenotype and function of plastic-adherent progenitor
cells in human hematopoietic tissue. Exp Hematol 23:817, 1995
(abstr)
19. Kerk DH, Henry EA, Eaves AC, Eaves CJ: Two classes of
primitive pluripotent hemopoietic progenitor cells: Separation by
adherence. J Cell Physiol 125:127, 1985
20. Bearpark AD, Gordon MY: Adhesive properties distinguish
subpopulations of haemopoietic stem cells with different spleen colony-forming andmarrow repopulating capacities. Bone Marrow
Transplant 4:625, 1989
4413
21. Kiefer F, Wagner EF, Keller G : Fractionation of mouse bone
marrow by adherence separates primitive hematopoietic stem cells
from in vitro colony-forming cells and spleen-forming cells. Blood
78:2577, 1991
22. Myers CD, Katz FE, Joshi G , Millar JL: A cell line secreting
stimulating factors for CFU-GEMM culture. Blood 64:152, 1984
23. Taswell C: Limiting dilution assays for the determination
of immunocompetent cell frequencies. I. Data analysis. J Immunol
126:1614, 1981
24. Scott MA,Ager S, Apperley JF, Jestice HK, Bloxham D,
Boraks P, Mahendra P, Marcus RE: Peripheral blood stem cell harvesting in multiple myeloma and malignant lymphoma. Leuk Lymph
19:479, 1995
25. Gordon MY, Lewis JL, Goldman JM: Influence of cultured
bone marrow-derived stromal cells on the proliferation activity of
primitive hematopoietic progenitor cells. Exp Hematol 23:784, 1995
(abstr)
26. Gordon MY, Lewis JL, Apperley JF, Goldman JM, Scott
MA, Marcus RE, Blackett NM: Primitive progenitor cells in normal
human bone marrow, peripheral blood progenitor cell harvests, umbilical cord blood and chronic myeloid leukaemia display kinetic
differences in renewaland generation of granulocyte-macrophage
colony-forming cells. Exp Hematol 22:789, 1994 (abstr)
27. Till JE, McCulloch EA, Siminovitch L: A stochastic model
of stem cell proliferation basedonthe
growth of spleen colony
forming cells. Proc Natl Acad Sci USA 51:29, 1964
28. Humphries RK, Eaves AC, Eaves CJ: Self-renewal of hemopoietic stem cells during mixed colony formation in vitro. Proc Natl
Acad Sci USA 78:3629, 1981
29.Bregni M, MagniM,Siena S, DiNicola M, Bonadonna G ,
Gianni A M : Humanperipheralbloodhematopoieticprogenitorsare
optimaltargets of retroviral-mediatedgenetransfer.Blood80:1418,
1992
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1995 86: 4468-4473
Plastic-adherent progenitor cells in mobilized peripheral blood
progenitor cell collections
MA Scott, JF Apperley, HK Jestice, DM Bloxham, RE Marcus and MY Gordon
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