|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 111-117
Stroma-Contact Prevents Loss of Hematopoietic Stem Cell Quality
During Ex Vivo Expansion of CD34+ Mobilized Peripheral
Blood Stem Cells
By
Dimitri A. Breems,
Ellen A.W. Blokland,
Karen E. Siebel,
Angelique
E.M. Mayen,
Lilian J.A. Engels, and
Rob E. Ploemacher
From the Institute of Hematology, Erasmus University Rotterdam,
Rotterdam, The Netherlands.
 |
ABSTRACT |
Stroma-supported long-term cultures (LTC) allow estimation of stem
cell quality by simultaneous enumeration of hematopoietic stem cell
(HSC) frequencies in a graft using the cobblestone area forming cell
(CAFC) assay, and the ability of the graft to generate progenitors in
flask LTC (LTC-CFC). We have recently observed that the number and
quality of mobilized peripheral blood stem cells (PBSC) was low in
patients having received multiple rounds of chemotherapy. Moreover,
grafts with low numbers of HSC and poor HSC quality had a high
probability to cause graft failure upon their autologous infusion.
Because ex vivo culture of stem cells has been suggested to present an
attractive tool to improve hematological recovery or reduce graft size,
we have studied the possibility that such propagation may affect stem
cell quality. In order to do so, we have assessed the recovery of
different stem cell subsets in CD34+ PBSC after a 7-day
serum-free liquid culture using CAFC and LTC-CFC assays. A numerical
expansion of stem cell subsets was observed in the presence of
interleukin-3 (IL-3), stem cell factor, and IL-6, while stroma-contact,
stromal soluble factors, or combined addition of FLT3-ligand and
thrombopoietin improved this parameter. In contrast, ex vivo culture
severely reduced the ability of the graft to produce progenitors in LTC
while stromal soluble factors partly abrogated this quality loss. The
best conservation of graft quality was observed when the PBSC were
cultured in stroma-contact. These data suggest that ex vivo propagation
of PBSC may allow numerical expansion of various stem cell subsets,
however, at the expense of their quality. In addition, cytokine-driven
PBSC cultures require stroma for optimal maintenance of graft quality.
| |
INTRODUCTION |
MOBILIZED PERIPHERAL blood stem cells
(PBSC) are increasingly used to restore the formation of blood cells
after high-dose chemotherapy for solid tumors and hematological
cancers.1-3 More recently, PBSC from cancer patients are
cultured ex vivo4-6 because expanded hematopoietic stem
cells (HSC) may possibly reduce the time to hematopoietic recovery
after their transplantation. Furthermore, the use of smaller
transplants may lead to a reduction of contaminating tumor cells.
Although clinical ex vivo expansion trials have already started, many
questions are still unanswered. Firstly, although many investigators
have shown that the total number of CD34+ cells,
progenitors, and primitive stem cells in PBSC can be expanded in
vitro,4-10 there has been no report of improved
hematopoietic recovery using such in vitro propagated
grafts.4-6 Secondly, because of the nonmyeloablative
conditioning regimens and/or cotransplantation of unmanipulated
HSC in these studies, it is also not apparent if primitive stem cells
are still capable of long-term engraftment after ex vivo culture. Ex
vivo propagation studies in mice have shown both loss of in vivo
engraftment11 and an increased ability of cultured cells to
repopulate irradiated hosts.12 Diminished engraftment in
vivo may result from a reduced ability of stem cells to home to the
bone marrow (BM). Indeed, we have recently shown that short incubations
of murine stem cells with several cytokine combinations diminish their
lodgement in hematopoietic organs and hence their ability to repopulate
the hematopoietic system of irradiated recipients.13 In
addition, loss of primitive stem cell quality may also lead to a
reduced in vivo repopulating ability. Previously, we have studied 47
mobilized PBSC harvests of 21 autologous transplantation cancer
patients and shown that poorly mobilized PBSC harvests contain a low
number of primitive HSC (cobblestone area forming cell [CAFC] week
6), and also produce less progenitors per primitive stem cell in
stroma-supported long-term cultures (LTC-CFC).14 This poor
primitive stem cell quality was related with the number of
cytoreductive pretreatment rounds administered to the patients. In
addition, we have observed low CAFC week 6 numbers and low primitive
stem cell quality in the original autologous transplant of patients
that failed to engraft within 6 months after
transplantation.15
It has been shown that primary stromal feeder layers and stromal cell
lines support the culture of HSC.16-19 In 2- or 5-week
cultures without exogenous cytokines, the Verfaillie group has shown
that primitive stem cell (LTC-initiating cell; LTC-IC) recovery and
colony-forming cell (CFC) production in LTC was improved when normal
BM-derived HSC were propagated in stroma-noncontact cultures as
compared to stroma-contact.20-23 This improvement was
explained by proliferation inhibition of CFC and LTC-IC during direct
stroma-contact, possibly via adhesion of the fibronectin receptor to
stroma.22,23 The stroma-noncontact cultures were further
improved by the addition of interleukin-3 (IL-3) and macrophage
inflammatory protein-1 (MIP-1) to the medium and simplified by
using stroma-conditioned medium (SCM) instead of stroma-noncontact
transwell inserts.24 However, Koller et al25,26
have shown that cytokine-driven LTC-IC expansion can only be achieved
with the use of a stromal feeder layer.
In contrast to studies on normal BM-derived HSC, only a limited number
of studies are dedicated to ex vivo expansion of LTC-IC or CAFC week 6
from clinically relevant mobilized PB from cancer patients. In 7- to
12-day static liquid cultures of CD34-selected mobilized PBSC, a
maintenance of LTC-IC or CAFC week 6 has been
reported.7,9,10 Two- to 20-fold expansion of LTC-IC or CAFC
week 6 from mobilized PBSC was only observed in cultures containing
stromal factors and/or accessory cells and in perfusion
bioreactors.8-10 However, those studies did not provide
information about the HSC quality.
In the present study we have focused on the effect of ex vivo
propagation on both the number and quality of HSC because, in our view,
numerical expansion of HSC can only be effective if their quality is
not reduced at the same time. We used CD34-selected mobilized PBSC from
myeloma and lymphoma patients in our experiments because this HSC
source is also used in most clinical ex vivo expansion
studies.4-6 In 7-day serum-free cultures supplemented with
IL-3, stem cell factor (SCF), and IL-6 with or without FLT3-ligand (FL)
and thrombopoietin (TPO), the effect of SCM addition from various
stromal cell lines was tested in comparison with direct contact with a
murine stromal cell layer and stroma-noncontact cultures on the
recovery of progenitors cell and primitive stem cell numbers and their
quality. The assessment of different HSC subsets was done using the
human CAFC assay wherein the CAFC week 2 to 4 are tentative indicators
of progenitor cell activity and transiently repopulating HSC, while
CAFC week 6 is interpreted as indicator of more primitive, long-term
repopulating stem cells.14,18 In parallel flask-LTC the CFC
production was determined in the corresponding weeks as an estimate of
total graft quality. LTC-CFC production and CAFC frequency allowed us
to assess the individual primitive stem cell quality.
| |
MATERIALS AND METHODS |
Mobilized PB.
Nine leukapheresis products from four patients with non-Hodgkin's
lymphoma, four with multiple myeloma, and one with Burkitt's lymphoma
in remission were used in this study. Before leukapheresis the HSC were
mobilized to the blood after several courses of chemotherapy using
granulocyte colony-stimulating factor (Filgrastim,
recombinant-methionyl human G-CSF; Roche, Mijdrecht, The Netherlands)
as described before.14 After cell collection, an excess of
erythrocytes was removed using buffy coat centrifugation. Fresh or
frozen and thawed leukaphereses were subjected to CD34-selection to
enrich for HSC. For CD34-selection the following methods were used
according to the guidelines of the suppliers: Ceprate SC column
(CellPro, Bothell, WA), Dynal CD34 progenitor cell selection system
(Dynal, Oslo, Norway), and MACS CD34 isolation kit (Miltenyi Biotec,
Bergisch Gladbach, Germany). Before CD34-selection using the Dynal
system and the MACS kit a density gradient was performed (1.077 g/mL,
Lymphoprep; Nycomed, Olso, Norway). After selection the percentage
CD34+ cells was determined as described
before.14 Table 1 shows the
frequency of the different stem cell subsets in the CD34+
selected PBSC before ex vivo culturing as determined using flow
cytometry, CFC, and CAFC assays.
Cytokines.
The following purified recombinant cytokines were kindly provided:
human granulocyte-macrophage-CSF (GM-CSF), human IL-6, and murine SCF
from Genetics Institute (Cambridge, MA); human FL, human G-CSF, and
human SCF from Amgen (Thousand Oaks, CA); human IL-3 from Gist Brocades
(Delft, The Netherlands); and human TPO from Genentech (South San
Francisco, CA).
Serum-free liquid culture.
Serum-free liquid culture experiments were performed in 35-mm bacterial
dishes (Greiner, Alphen a/d Rijn, The Netherlands) to prevent strong
adherence of the hematopoietic cells to the plastic surface. The
serum-free Iscove's modified Dulbecco's medium with Glutamax-1 (IMDM;
GIBCO, Breda, The Netherlands) contained 1% bovine serum albumin
(A9418; Sigma, St Louis, MO), penicillin (100 U/mL; GIBCO),
streptomycin (100 µg/mL; GIBCO), 10 4 mol/L
 -mercaptoethanol (Merck, Darmstadt, Germany), bovine insulin (10
µg/mL; GIBCO), 15 µmol/L cholesterol (Sigma), 15 µmol/L linolic
acid (Merck), iron-satured human transferrin (0.62 g/L; Intergen,
Purchase, NY), cytidine (1 µg/mL; Sigma), adenosine (1 µg/mL;
Sigma), uridine (1 µg/mL; Sigma), guanosine (1 µg/mL; Sigma),
thymidine (1 µg/mL; Sigma), 2 -deoxycytidine (1 µg/mL;
Sigma), 2-deoxyadenosine (1 µg/mL; Sigma),
2-deoxyguanosine (1 µg/mL; Sigma). Forty thousand
CD34+ PBSC in 2 mL serum-free medium supplemented with IL-3
(25 ng/mL), human SCF (100 ng/mL), IL-6 (100 ng/mL), and
anti-human-transforming growth factor-1 (TGF1; 1.0 µg/mL;
R&D Systems, Abingdon, UK) with or without FL (100 ng/mL) and TPO (10
ng/mL) were cultured at 37°C and 10% CO2. After 7 days
of culture, the cells were collected from the dishes after scraping
with a cell scraper (Greiner) and rinsing with IMDM. After washing, the
cells were resuspended in IMDM and plated in CFC, CAFC, and flask-LTC
assays.
Stroma-conditioned media.
Confluent layers were grown of the stromal cell lines FBMD-1, L87/4,
and L88/5.18,19,27 The cells were cultured in IMDM
supplemented with 10% fetal calf serum (FCS; Summit, Fort Collins,
CO), penicillin (100 U/mL), streptomycin (100 µg/mL), and
104 M -mercaptoethanol. The FBMD-1 cells were
maintained at 33°C and 10% CO2 and the L87/4 and L88/5
cells at 37°C and 10% CO2. When the layers were
confluent, the medium was removed and rinsed twice with IMDM.
Serum-free medium was added to the confluent stromal layers and
conditioned for 7 days. The SCM were harvested, the nonadherent (NA)
cells were removed by centrifugation, and the media were stored at
 20°C until use. Control medium was prepared by parallel
incubations without the stromal cell lines. In the cultures, 50% SCM
or control medium was used.
Stroma-contact cultures.
In 35-mm tissue culture dishes (Falcon, Franklin Lakes, NJ) confluent
layers were grown of the stromal cell line FBMD-1. When the layers were
confluent, 40,000 CD34+ PBSC were cultured on these stromal
feeders in the same medium and under the same conditions as used for
the serum-free liquid cultures. After 7 days of culture, the NA cells
were collected from the dish and after two rinses with IMDM replaced by
1 mL of 0.1% trypsin (GIBCO) for 5 minutes. The digestion was stopped
by adding 1 mL of ice-cold FCS and the dish was scraped with a cell
scraper to include all adherent cells. The NA and adherent cells were
pooled and after washing the cells were resuspended in IMDM and plated
in CFC, CAFC, and flask-LTC assays.
Stroma-noncontact cultures.
In 6-well plates (Costar, Cambridge, MA) confluent layers were grown of
the stromal cell line FBMD-1. When the layers was confluent, 40,000
CD34+ PBSC were cultured in a collagen-coated membrane
transwell insert (0.4 µm pore size; Costar) placed above the FBMD-1
stromal layer in the same medium and under the same conditions as used
for the serum-free liquid cultures. After 7 days of culture, all cells
were collected from the transwell insert and after washing the cells
were resuspended in IMDM and plated in CFC, CAFC, and flask-LTC assays.
Colony-forming cell assay.
Quantification of the number of colony-forming units
granulocyte/macrophage (CFU-GM) and burst-forming units erythroid
(BFU-E) was performed using a semisolid CFC assay containing
erythropoietin (Boehringer, Mannheim, Germany), G-CSF, GM-CSF, IL-3,
and murine SCF as described before.14 CFU-GM and BFU-E were
counted on day 14 of culture in the same dish.
Long-term cultures in flasks.
Confluent stromal layers of FBMD-1 cells in 25-cm2 flasks
(Costar) were overlaid with 30,000 CD34+ PBSC or the output
of 30,000 cultured CD34+ PBSC. The cells were cultured in
IMDM supplemented with 10% FCS, 5% horse serum (Integro, Zaandam, The
Netherlands), 105 mol/L hydrocortisone
21-hemisuccinate (Sigma), penicillin (100 U/mL), streptomycin (100
µg/mL), and 104 mol/L -mercaptoethanol. IL-3
(10 ng/mL) and G-CSF (20 ng/mL) were added weekly to the cultures.
Flask cultures were set up in duplicate and maintained at 33°C and
10% CO2 for 6 weeks with weekly half-medium changes and
therefore removal of half of the NA cells. The NA-CFC output of
individual flask cultures was determined on weeks 2, 4, and 6 and was
not corrected for the weekly demi-depopulations. At the end of 6 weeks
the number of CFC in the adherent layer was also determined. To this
purpose, the NA cells were collected from the flasks and after two
rinses with IMDM replaced by 3 mL of 0.1% trypsin for 5 minutes. The
digestion was stopped by adding 1 mL of ice-cold FCS and the flasks
were scraped with a cell scraper to include all adherent cells. A
single cell suspension was made by sieving the cell suspension through
a 100-µm nylon filter. The cell suspension was taken up in IMDM and
several concentrations of the cell suspension were plated in a
semisolid CFC assay.
CAFC assay.
Limiting dilution CAFC assays were performed on confluent stromal
layers of FBMD-1 cells in flat-bottom 96-well plates (Falcon). The
cultures were maintained under the same conditions as the LTC in
flasks. CD34+ PBSC were overlaid in a limiting dilution
setup. Twelve dilutions twofold apart were used for each sample with 15
replicate wells per dilution. The percentage of wells with at least one
phase-dark hematopoietic clone of at least five cells (ie, cobblestone
area) beneath the stromal layer was determined at weeks 2, 4, and 6 and
CAFC frequencies were calculated using Poisson statistics.
Statistical analysis.
Data were analyzed using GraphPad Instat (GraphPad Software, San Diego,
CA). The means of two populations were compared using a paired
Student's t-test.
| |
RESULTS |
Expansion of progenitor and primitive stem cells in liquid cultures.
In 7-day serum-free liquid cultures containing IL-3, SCF, IL-6, and
TGF1 (3/S/6/T), we were able to expand progenitor cells (CFC
and CAFC week 2 to 4) 2.6-fold and to maintain primitive stem cells
(CAFC week 6: 1.2-fold expansion) (Fig
1A). As reported recently, the expansion of
HSC could significantly be improved when SCM from the FBMD-1, L87/4, or
L88/5 stromal cell lines was added to the liquid
cultures.10 Using L88/5 SCM, progenitor cells and primitive
stem cells were 7.9-fold and 3.5-fold expanded, respectively (Fig 1A).
View larger version (43K):
[in this window]
[in a new window]
| Fig 1.
The effect of 7-day serum-free liquid cultures containing
IL-3, SCF, IL-6, and TGF1 with or without stroma-conditioned
media (SCM) on (A) the expansion of different stem cell subsets (CFC
and CAFC weeks 2 to 6) and (B) the ability of stem cells to produce NA
CFC in parallel flask long-term cultures (LTC) for 6 weeks. At week 6
the stroma-adherent (SA) CFC content was also determined. Comparison
between no SCM and with SCM: *, P < .05. ( ), No SCM (n =
7 to 9); ( ), FBMD-1 SCM (n = 7 to 9); ( ), L87/4 SCM (n = 3 to
5); ( ), L88/5 SCM (n = 3 to 5).
|
|
Graft quality of unexpanded CD34+ mobilized PBSC.
To determine the in vitro graft quality of unexpanded CD34+
PBSC, flask-LTC were performed in parallel to the CAFC assay. In
stroma-dependent flask-LTC the ability to produce NA-CFC was measured
at weeks 2, 4, and 6. At week 6 the number of stroma-adherent (SA) CFC
was also assessed. Throughout the culture period the total NA-CFC
production ranged between 21 and 36 per 100 CD34+ input
cells and at week 6 the adherent layer still contained 12 CFC per 100
input cells (Table 2). These results show
that unmanipulated CD34+ PBSC were able to produce a
relatively constant number of progenitors for at least 6 weeks of
culture.
Loss of graft quality of expanded CD34+ mobilized PBSC
after liquid culture.
Using the same setup for liquid cultures as described above, the in
vitro graft quality of CD34+ PBSC was determined after a
7-day serum-free liquid culture. In contrast to the expansion of HSC
numbers (Fig 1A), the ability of stem cells to produce LTC-CFC was
diminished as compared to the input cells (Fig 1B). CD34+
PBSC that had been cultured in the presence of 3/S/6/T produced 80%
NA-CFC at week 2 as compared with unexpanded CD34+ PBSC. At
later weeks there was a further reduction of NA-CFC production (week 4,
31%; week 6, 13%). Also, at week 6 the SA-CFC content was only a
fraction of the control CD34+ PBSC (13%). The addition of
FBMD-1 and L88/5 SCM showed only a modest improvement of the graft
quality (NA-CFC week 2, 100% and 88%; NA-CFC week 4, 51% and 42%;
NA-CFC week 6, 16% and 14%; SA-CFC week 6, 27% and 34%,
respectively, as compared with unexpanded cells). L87/4 SCM did not
influence the graft quality.
Expansion of progenitor and primitive stem cells in stroma-contact
and stroma-noncontact cultures.
To investigate whether direct contact with stroma cells would similarly
improve the recovery and quality of primitive stem cells as did soluble
stromal factors, we studied the fate of HSC when cultured for 7 days in
serum-free medium containing 3/S/6/T in FBMD-1 SCM, in direct
contact with a FBMD-1 stromal layer and in FBMD-1 stroma-noncontact
(Fig 2A). SCM and stroma-contact showed
no significant differences in their effect of the numerical expansion
of progenitors and primitive stem cells, and both significantly
improved generation of progenitor cells (CFC and CAFC weeks 2 to 4) as
compared with stroma-noncontact cultures.
View larger version (40K):
[in this window]
[in a new window]
| Fig 2.
The effect of FBMD-1 SCM, FBMD-1 stroma-contact, and
FBMD-1 stroma-noncontact on (A) the expansion of different stem cell
subsets (CFC and CAFC week 2 to 6) and (B) the ability of stem cells to
produce NA CFC in parallel flask LTC for 6 weeks in 7-day serum-free
liquid cultures containing IL-3, SCF, IL-6, and TGF1. At week 6
the stroma-adherent (SA) CFC content also was determined. Comparison
between FBMD-1 SCM and FBMD-1 stroma-contact or stroma-noncontact:
°, P < .05. Comparison between FBMD-1 stroma-noncontact
and FBMD-1 stroma-contact:  , P < .05. (),
SCM (n = 5 to 6); (), stroma-contact (n = 5 to 6); (),
stroma-noncontact (n = 3).
|
|
Stroma-contact prevents loss of primitive stem cell quality in
expansion cultures.
CD34+ PBSC cultured in FBMD-1 stroma-contact showed a
significantly improved graft quality as compared with FBMD-1 SCM and
stroma-noncontact cultures (Fig 2B). At weeks 2 and 4 the
stroma-contact expanded CD34+ PBSC produced 143% and 91%
NA-CFC, respectively, as compared with the input CD34+
PBSC. The NA-CFC and SA-CFC at week 6 of stroma-contact expanded cells
were still 60% and 73%, respectively, as compared with unexpanded
cells.
In Table 3 the primitive stem cell quality
in week 6 LTC is summarized. In 7-day serum-free liquid cultures
containing 3/S/6/T there was a dramatic loss of graft quality (13%
of input) as expressed in NA + SA LTC-CFC at week 6 (Table 3, second
column).The addition of FBMD-1 SCM could only slightly prevent this
quality loss (21% v 13%). In FBMD-1 stroma-noncontact
cultures quality of primitive stem cells was more preserved (38%
v 13%), while FBMD-1 stroma-contact cultures proved to be the
best in preventing the loss of graft quality (66% v 13%). By
calculating the mean number of LTC-CFC produced in week 6 flask-LTC per
CAFC week 6, we were able to estimate the average individual primitive
stem cell quality of expanded CD34+ PBSC (Table 3, third
column). Although the CD34+ PBSC that had been propagated
in FBMD-1 stroma-contact had also the best average LTC-CFC per CAFC at
week 6, in all culture conditions there was extensive loss (6- to
13-fold) of individual primitive stem cell quality as compared with
unexpanded CD34+ PBSC.
View this table:
[in this window]
[in a new window]
|
Table 3.
CAFC Percentage, NA + SA LTC-CFC Production, and
Mean LTC-CFC per CAFC at Week 6 of Mobilized PBSC Before and After
Ex Vivo Expansion
|
|
FL and TPO further improve the numerical expansion and quality
maintenance of both progenitor and primitive stem cells.
To test whether the effects of stroma and stroma-elaborated activities
was due to the recently cloned cytokines FL and TPO, we performed
experiments in which FL and TPO were added to the 7-day cultures in the
presence of 3/S/6/T with or without FBMD-1 SCM or FBMD-1
stroma-contact. The combination of IL-3, SCF, IL-6, TGF1, FL, and
TPO with FBMD-1 stroma-contact led to a 21.1- and 4.9-fold expansion of
CAFC weeks 2 and 6, respectively (Table 4).
Addition of FL/TPO to 7-day FBMD-1 stroma-contact cultures also further
enhanced the nonadherent CFC production in LTC leading to a complete
maintenance of LTC-CFC quality (Table 5).
As a result, inclusion of FL/TPO improved the recovery of all
progenitor and stem cell subsets tested, and their ability to generate
CFC. Remarkably, the inclusion of FBMD-1 SCM or FBMD-1 stroma-contact
still gave further improvement of these parameters, indicating that the
stroma-related effects described here were not mediated by FL
and/or TPO.
View this table:
[in this window]
[in a new window]
|
Table 4.
CFC and CAFC Week-Type Expansion of Mobilized PBSC After
Ex Vivo Expansion With or Without the Addition of FL and TPO
|
|
View this table:
[in this window]
[in a new window]
|
Table 5.
Nonadherent LTC-CFC Production of Mobilized PBSC After
Ex Vivo Expansion With or Without the Addition of FL and TPO
|
|
| |
DISCUSSION |
At least three different groups have reported the transplantation of
CD34-selected mobilized PBSC after a period of in vitro culture in an
attempt to expand repopulating cell numbers.4-6 Using 8- to
12-day liquid culture systems with various cytokine combinations, 28
patients were transplanted with ex vivo propagated CD34+
cells. These studies showed an up to 332-fold expansion of progenitor
cells in the PBSC transplants; however, the expanded grafts did not
significantly improve hematologic recovery. This suggests that a
numerical expansion of progenitors may not be of relevance for
short-term hematopoietic recovery posttransplantation. Indeed, this
notion is supported by a murine study showing that committed
progenitors play no role in short-term in vivo
repopulation.28
In the present study we investigated the recovery of both progenitors
and primitive stem cells in CD34+ PBSC after ex vivo
propagation in more detail. We showed an expansion of both progenitor
and primitive stem cell numbers, which was consistent with previous
reports.4-10 As reported before, the effect of soluble
stromal factors significantly improved this expansion.10
Although we found an expansion of CFC and various CAFC subsets in these
cultures, the grafts' ability to generate progenitors was only
maintained in week 2 flask-LTC, while the cultured cells produced
dramatically less progenitors at later weeks. This showed that an
absolute numerical expansion of progenitors and primitive stem cells
may have occurred at the expense of the ability of primitive HSC to
generate CFC in long-term stroma-supported cultures, or alternatively,
that not all primitive HSC survived. This situation could explain the
inability of expanded grafts to improve hematologic recovery in
conditioned recipients.
Although the clinical relevance of a reduced ability of expanded
primitive stem cells to produce CFC at later weeks is not fully clear,
we believe that this observation is more than just "an in vitro
artifact." Our group has recently observed that autologous grafts
that were unable to lead to significant hematopoietic repopulation of
patients at 3 months posttransplantation contained low CAFC week 6
numbers, while their ability to generate CFC in LTC was very
low.15 In addition, we have reported that only few CAFC
week 6 are mobilized in patients that have received intense cytotoxic
chemotherapy, and that their quality is reduced.14
In the second part of this study we have investigated factors that
could improve the expansion of primitive stem cells (CAFC week 6) and
maintain their quality (LTC-CFC at week 6). We have found that both
soluble stromal factors and stroma-contact increase the expansion of
CAFC week 6 in the presence of cytokines (IL-3/SCF/IL-6) and
TGF1. The improved generation of CAFC week 6 is probably the
combined result of their proliferation and conservation. The Verfaillie
group has shown that the maintenance or expansion of LTC-IC in 2-week
stroma-noncontact cultures supplemented with cytokines (IL-3/MIP-1)
is the result of extensive proliferation of a small fraction of the
input LTC-IC.29 In addition, the same research group using
2- or 5-week stroma-noncontact cultures without addition of cytokines
has reported an inhibitory effect of stroma-contact on CFC and LTC-IC
proliferation as compared with stroma-noncontact
cultures.22,23 In our study we do not observe inhibitory
effects of stroma-contact on CAFC week 6 expansion. This can be
explained by the addition of cytokines (IL-3/SCF/IL-6) to both
stroma-contact and stroma-noncontact cultures in our experiments, which
may have overruled this stroma-contact mediated proliferation
block.23 Furthermore, the addition of neutralizing
antibodies directed against TGF1 could have further abrogated the
proliferation inhibition of stroma-contact, because TGF1 is an
important inhibitor of primitive stem cell proliferation30
and is produced by the FBMD-1 stromal cell line.18 In
addition to the favorable effect of stroma-contact for numerical CAFC
expansion, direct stroma-contact appears to be required for
conservation of total graft quality (LTC-CFC week 6). Because soluble
stromal factors only partly protect against quality loss of HSC, it may
be argued that the concentration of SCM in our cultures may have been
too low; however, stroma-noncontact cultures gave comparable results as
SCM-containing cultures. As a result, our data support the observations
of Koller et al25,26 in that stromal cells exert a
favorable effect on BM-derived LTC-IC expansion in 2-week cultures
containing IL-3, SCF, GM-CSF, and erythropoietin. In the light of the
recent observations from other investigators that in vitro expanded
progenitor and primitive stem cell grafts do not improve time to
hematologic recovery, it seems indeed pertinent to include stromal
elements in bioreactors for cytokine-driven ex vivo expansion of HSC
contained in mobilized PB.31
A recent study of Petzer et al32 reports that in 10-day
liquid cultures IL-3, SCF, FL, and TPO are the most important cytokines
for the expansion of LTC-IC from
CD34+/CD38 BM cells. In addition, it has
been shown that in these cultures there is no loss of CFC-producing
ability.33 Therefore, we performed additional experiments
in which FL and TPO were added to the expansion cultures. Indeed, the
addition of FL/TPO to expansion cultures improved the recovery of
progenitors and primitive stem cells and that this FL/TPO effect was
still present in the presence of soluble stromal factors and direct
stromal contact. In addition, FL/TPO together with stromal factors
further improved the LTC-CFC production at week 6 resulting in a
complete maintenance of primitive stem cell quality. These observations
strongly support the addition of FL and TPO to stroma-dependent
expansion strategies.
| |
FOOTNOTES |
Submitted May 13, 1997;
accepted September 2, 1997.
Address reprint requests to Rob E. Ploemacher, PhD, Institute of
Hematology, Erasmus University Rotterdam, PO Box 1738, 3000 DR
Rotterdam, The Netherlands.
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 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors gratefully acknowledge Prof Dr Bob Löwenberg for
providing continuous support. We thank Drs Jan Cornelissen and Martin
Schipperus for providing the patient material.
| |
REFERENCES |
1.
Körbling M,
Dörken 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
67:529,
1986[Abstract]
2.
Kessinger A,
Armitage JO,
Landmark JD,
Weisenburger DD:
Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells.
Exp Hematol
14:192,
1986[Medline]
[Order article via Infotrieve]
3.
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[Medline]
[Order article via Infotrieve]
4.
Brugger W,
Heimfeld S,
Berenson RJ,
Mertelsmann R,
Kanz L:
Reconstitution of hematopoiesis after high-dose chemotherapy by autologous progenitor cells generated ex vivo.
N Eng J Med
333:283,
1995[Abstract/Free Full Text]
5.
Williams SF,
Lee WJ,
Bender JG,
Zimmerman T,
Swinney P,
Blake M,
Carreon J,
Schilling M,
Smith S,
Williams DE,
Oldham F,
Van Epps D:
Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer.
Blood
87:1687,
1996[Abstract/Free Full Text]
6.
Alcorn MJ,
Holyoake TL,
Richmond L,
Pearson C,
Farrell E,
Kyle B,
Dunlop DJ,
Ritzsimons E,
Stewart WP,
Pragnell IB,
Franklin IM:
CD34+ cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity.
J Clin Oncol
14:1839,
1996[Abstract]
7.
Henschler R,
Brugger W,
Luft T,
Frey T,
Mertelsmann R,
Kanz L:
Maintenance of transplantation potential in ex vivo expanded CD34+-selected human peripheral blood progenitor cells.
Blood
84:2898,
1994[Abstract/Free Full Text]
8.
Van Zant G,
Rummel SA,
Koller MR,
Larson DB,
Drubachevsky I,
Palsson M,
Emerson SG:
Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood.
Blood Cells
20:482,
1994[Medline]
[Order article via Infotrieve]
9.
Sandstrom CE,
Bender JG,
Papoutsakis ET,
Miller WM:
Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells.
Blood
86:958,
1995[Abstract/Free Full Text]
10.
Breems DA,
Blokland EAW,
Ploemacher RE:
Stroma-conditioned media improve expansion of human primitive hematopoietic stem cells and progenitor cells.
Leukemia
11:142,
1997[Medline]
[Order article via Infotrieve]
11.
Peters SO,
Kittler ELW,
Ramshaw HS,
Quesenberry PJ:
Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts.
Blood
87:30,
1996[Abstract/Free Full Text]
12.
Holyoake TL,
Freshney MG,
McNair L,
Parker AN,
McKay PJ,
Steward WP,
Fitzsimons E,
Graham GJ,
Pragnell IB:
Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow.
Blood
87:4589,
1996[Abstract/Free Full Text]
13.
Van der Loo JCM,
Ploemacher RE:
Marrow- and spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are decreased by preincubation with hematopoietic growth factors.
Blood
85:2598,
1995[Abstract/Free Full Text]
14.
Breems DA,
Van Hennik PB,
Ku adasi N,
Boudewijn A,
Cornelissen JJ,
Sonneveld P,
Ploemacher RE:
Individual stem cell quality in leukapheresis products is related to the number of mobilized stem cells.
Blood
87:5370,
1996[Abstract/Free Full Text]
15. (abstr)
Van Hennik PB,
Breems DA,
Withagen CM,
Slaper ICM,
Ploemacher RE:
Graft-failure can be predicted by testing the ability of stem cells to produce progenitors in long-term stroma-supported cultures.
Exp Hematol
25:742,
1997
16.
Dexter TM,
Allen TD,
Lajtha LG:
Conditions controlling the proliferation of haemopoietic stem cells in vitro.
J Cell Physiol
91:335,
1977[Medline]
[Order article via Infotrieve]
17.
Gartner S,
Kaplan HS:
Long-term culture of human bone marrow cells.
Proc Natl Acad Sci USA
77:4756,
1980[Medline]
[Order article via Infotrieve]
18.
Breems DA,
Blokland EAW,
Neben S,
Ploemacher RE:
Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay.
Leukemia
8:1095,
1994[Medline]
[Order article via Infotrieve]
19.
Thalmeier K,
Meissner P,
Reisbach G,
Falk M,
Brechtel A,
Dörmer P:
Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity.
Blood
83:1799,
1994[Abstract/Free Full Text]
20.
Verfaillie CM:
Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis.
Blood
79:2821,
1992[Abstract]
21.
Verfaillie CM:
Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation.
Blood
82:2045,
1993[Abstract]
22.
Hurley RW,
McCarthy JB,
Verfaillie CM:
Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation.
J Clin Invest
96:511,
1995[Medline]
[Order article via Infotrieve]
23.
Verfaillie CM,
Catanzaro P:
Direct contact with stroma inhibits proliferation of human long-term culture initiating cells.
Leukemia
10:498,
1996[Medline]
[Order article via Infotrieve]
24.
Verfaillie CM,
Catanzarro PM,
Li W-N:
Macrophage inflammatory protein 1, interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro.
J Exp Med
179:643,
1994[Abstract]
25.
Koller MR,
Palsson MA,
Manchel I,
Palsson BØ:
Long-term culture-initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects.
Blood
86:1784,
1995[Abstract/Free Full Text]
26.
Koller MR,
Manchel I,
Brott DA,
Palsson BØ:
Donor-to-donor variability in the expansion potential of human bone marrow cells is reduced by accessory cells but not by soluble growth factors.
Exp Hematol
24:1484,
1996[Medline]
[Order article via Infotrieve]
27.
Thalmeier K,
Meissner P,
Reisbach G,
Hültner L,
Mortensen BT,
Brechtel A,
Oostendorp RAJ,
Dörmer P:
Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines.
Exp Hematol
24:1,
1996[Medline]
[Order article via Infotrieve]
28. Zijlmans JMJM, Kleiverda K, Heemskerk DPM, Kluin PhM, Visser
JWM, Willemze R, Fibbe WE: No role for committed progenitor cells in
engraftment following transplantation of murine cytokine-mobilized
blood cells. Blood 86:112a, 1995 (abstr, suppl 1)
29.
Verfaillie CM,
Miller JS:
A novel single-cell proliferation assay shows that long-term culture-initiating cell (LTC-IC) maintenance over time results from the extensive proliferation of a small fraction of LTC-IC.
Blood
86:2137,
1995[Abstract/Free Full Text]
30.
Cashman JD,
Eaves AC,
Raines EW,
Ross R,
Eaves CJ:
Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-.
Blood
75:96,
1990[Abstract]
31.
Koller MR,
Bender JG,
Miller WM,
Papoutsakis ET:
Expansion of primitive human hematopoietic progenitors in perfusion bioreactor system with IL-3, IL-6 and stem cell factor.
Biotechnology
11:358,
1993[Medline]
[Order article via Infotrieve]
32.
Petzer AL,
Zandstra PW,
Piret JM,
Eaves CJ:
Differential cytokine effects on primitive (CD34+CD38) human hematopoietic cells: Novel responses to Flt3-ligand and thrombopoietin.
J Exp Med
183:2551,
1996[Abstract]
33.
Petzer AL,
Hogge DE,
Lansdorp PM,
Reid DS,
Eaves CJ:
Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.
Proc Natl Acad Sci USA
93:1470,
1996[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S. Gottschling, R. Saffrich, A. Seckinger, U. Krause, K. Horsch, K. Miesala, and A. D. Ho
Human Mesenchymal Stromal Cells Regulate Initial Self-Renewing Divisions of Hematopoietic Progenitor Cells by a {beta}1-Integrin-Dependent Mechanism
Stem Cells,
March 1, 2007;
25(3):
798 - 806.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Vanheusden, S. Van Coppernolle, M. De Smedt, J. Plum, and B. Vandekerckhove
In Vitro Expanded Cells Contributing to Rapid Severe Combined Immunodeficient Repopulation Activity Are CD34+38-33+90+45RA-
Stem Cells,
January 1, 2007;
25(1):
107 - 114.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Chen, H. Xu, C. Wan, M. McCaigue, and G. Li
Bioreactor Expansion of Human Adult Bone Marrow-Derived Mesenchymal Stem Cells
Stem Cells,
September 1, 2006;
24(9):
2052 - 2059.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kobune, Y. Ito, Y. Kawano, K. Sasaki, H. Uchida, K. Nakamura, H. Dehari, H. Chiba, R. Takimoto, T. Matsunaga, et al.
Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-{beta}2m-/- repopulating cells.
Blood,
August 15, 2004;
104(4):
1002 - 1009.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Harvey and E. Dzierzak
Cell-Cell Contact and Anatomical Compatibility in Stromal Cell-Mediated HSC Support During Development
Stem Cells,
May 1, 2004;
22(3):
253 - 258.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Chute, G. Muramoto, J. Fung, and C. Oxford
Quantitative Analysis Demonstrates Expansion of SCID-Repopulating Cells and Increased Engraftment Capacity in Human Cord Blood Following Ex Vivo Culture with Human Brain Endothelial Cells
Stem Cells,
March 1, 2004;
22(2):
202 - 215.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Mulloy, J. Cammenga, F. J. Berguido, K. Wu, P. Zhou, R. L. Comenzo, S. Jhanwar, M. A. S. Moore, and S. D. Nimer
Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element
Blood,
December 15, 2003;
102(13):
4369 - 4376.
[Abstract]
| |
Abstract
Introduction
Abstract