Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1199-1200
CORRESPONDENCE
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To the Editor: |
A potential role for leukemia inhibitory factor in the increased
clonogenicity of human fetal progenitor cells
A recent report by Shih et al demonstrates the ex vivo
expansion of human fetal CD34+, thy-1+
hematopoietic progenitors in the presence of a leukemia inhibitory factor (LIF)-induced, murine stromal cell-derived
factor.1 The authors show that expanded progenitor cells
retain their immature immunophenotype in vitro and that the engraftment
capacity in murine transplantation models is preserved. In this and
their previous report, they allude to the potential utility of such an
expansion promoting factor (SCEPF) in the clinical arena, including the
transplantation of stem cells from cord blood.1, 2
In their experiments, a given LIF concentration of 10 ng/mL results in
the production of a LIF-induced expansion promoting factor by the
murine stromal cell line AC 6.21. They do not report how this
concentration was determined to be optimal, nor do they report whether
a dose response relationship between LIF concentration in AC 6.21 stromal cell culture and the magnitude of expansion was observed. The
experimental design did not seem to address a potential direct effect
of human LIF, in the dose range used, on CD34+,
thy-1+ progenitors in culture, as control studies with
LIF-neutralizing antibody were not conducted.
Based on our own experiments, levels of LIF protein in unstimulated
adult peripheral (n = 6) and neonatal umbilical cord blood (n = 13)
are at the limit of detection when measured by enzyme linked
immunoadsorbant assay (ELISA) and are, therefore, several orders of
magnitude below the concentration used by the authors. A significant
rise in LIF protein levels, however, can be effected by stimulation of
mononuclear cells, especially those derived from cord blood. Combined
activation with anti-CD3 and IL-2 leads to a more than 20-fold increase
in LIF production by cord blood mononuclear cells, compared to a 7-fold
increase seen with adult PBMC (Figure).
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Levels of LIF protein measured by ELISA.
Stimulation with mouse anti-CD3 monoclonal antibody and interleukin-2
was carried out in liquid culture over 72 hours.
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This effect is blunted (4- and 5-fold increase, respectively) after
monocyte (CD14) depletion, suggesting that T cell-monocyte interaction
is required. Stimulation with interferon 
, tumor necrosis factor
, interleukin-1
, or lipopolysacharide results in moderate 2- to
3-fold increased LIF production by cord blood derived mononuclear cells.
Other groups have demonstrated the markedly increased clonogenicity of
cord blood-derived progenitors and its expansion potential upon
addition of LIF to culture conditions.3, 4 At least in the
murine model, LIF-induced expansion of progenitors appears to be based
on the increased production of more primitive progenitors with greater
replicative potential.5
We therefore wish to suggest that the expansion effect described
by Shih et al may rely on the supraphysiologic levels of LIF in their
stromal cell culture system that in turn lead to the elaboration of an
expansion promoting activity. Moreover, it remains to be determined,
whether in addition to the induction of SCEPF in murine stroma, human
LIF used at nanogram concentrations has an independent effect on the
expansion of human fetal progenitors. Taken together, the augmented
production of LIF by cord blood mononuclear cells may directly and/or
indirectly underly the increased in vitro and in vivo clonogenicity of
cord blood, compared to adult PBMC.
Peter Kurre
Fred Hutchinson Cancer Research Center and the Department of
Pediatrics
University of Washington
Seattle, WA
Stefan Burdach
Department of Pediatrics and BioCenter
University of
Halle-Wittenberg
Halle, Germany
| |
Footnotes |
Supported by grants from the Deutsche Krebshilfe (W
11/94 Bu2) and the Elterninitiative Kinderkrebsklinik eV.
| |
References |
1.
Shih CC, Hu MC, Hu J, et al.
A secreted and LIF-mediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells.
Blood.
2000;95:957-966.
2.
Shih CC, Hu MC, Hu J, Medeiros J, Forman SJ.
Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells.
Blood.
1999;94:1623-1636[Abstract/Free Full Text].
3.
Broxmeyer HE, Hangoc G, Cooper S, et al.
Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults.
Proc Natl Acad Sci U S A.
1992;89:4109-4113[Abstract/Free Full Text].
4.
Gabutti V, Timeus F, Ramenghi U, et al.
Expansion of cord blood progenitors and use for hemopoietic reconstitution.
Stem Cells.
1993;11(suppl 2):105-112[Abstract].
5.
Imamura M, Zhu X, Han M, et al.
In vitro expansion of murine hematopoietic progenitor cells by leukemia inhibitory factor, stem cell factor, and interleukin-1 beta.
Exp Hematol.
1996;24:1280-1288[Medline]
[Order article via Infotrieve]
| |
Response: |
Leukemia inhibitory factor does not have a direct effect on the
expansion of transplantable human hematopoietic stem cells
We thank Drs Kurre and Burdach for their interesting comments
about the possibility that leukemia inhibitory factor (LIF) might have
a direct effect on the expansion of human fetal progenitor cells. We
would like to take this opportunity to address those concerns that have
been raised by Drs Kurre and Burdach:
1. Whether a dose response relationship between LIF concentration
in AC6.21 stromal cell culture and the magnitude of expansion was
observed. Before we began to collect hundreds of liters of stromal conditioned medium from LIF-treated cultures (SCM-LIF) for protein purification, we asked ourselves whether the amount of LIF
in the stromal cell culture could be reduced without losing the
stem-cell-expansion promoting factor (SCEPF) activity. Experiments were
performed in our laboratory to determine the minimal concentration of
LIF for the production of SCEPF. SCM-LIFs were prepared from stromal
cell cultures treated with various amounts of LIF and assayed for SCEPF
activity as previously described.1,2 Our results
demonstrate that there is a dose response relationship between LIF
concentration in AC6.21 stromal cell culture and the magnitude of
expansion (Table).
There is no detectable
SCEPF activity in the SCM-LIFs when the stromal cell cultures were
treated with less than 50 pg/mL of LIF. SCEPF activity in the SCM-LIFs
is proportionally increased when the concentration of LIF in the
stromal cell cultures is increased from 50 pg/mL to 500 pg/mL. The
SCEPF activity in the SCM-LIFs is saturated when the concentration of
LIF is higher than 500 pg/mL. We now routinely use 500 pg/mL as
the optimal dose of LIF in our stromal-based culture system
and for preparation of SCM-LIFs for SCEPF protein purification.
2. Whether there is a potential direct effect of human LIF, in the
dose range used (10 ng/mL), on CD34+
thy-1+ progenitors in culture. We
have 3 sets of data that argue against the notion that LIF might have a
direct effect on CD34+ thy-1+ progenitors.
First, as we have shown in a previous report,1 there is no
significant difference in the ability to support proliferation and
differentiation of CD34+ thy-1+
progenitors between the stromal cultures treated with either 10 ng/mL
of human LIF or murine LIF, the latter of which cannot bind to the human LIF receptor. This result demonstrates that the
action of LIF is indirect and mediated via the stromal cells to
facilitate ex vivo expansion of transplantable human fetal BM
CD34+ thy-1+ cells. Since CD34+
thy-1+ cells respond very similarly to both human LIF
(which can bind to human and mouse LIF receptor) and murine LIF,
suggesting that human LIF does not directly have any significant effect
on CD34+ thy-1+ cells proliferation and
differentiation. Second, as we have shown in another
report,2 cells with CD34+
thy-1+ phenotype can be detected only in the positive
control culture (treated with 200% SCM-LIF) and not in any other
culture conditions treated with 200% stromal conditioned medium from
cultures in the absence of LIF (SCM), 10 ng/mL LIF, and various
combinations of cytokines including interleukin-3, interleukin-6,
granulocyte macrophage colony-stimulating factor, stem cell factor,
flt-3 ligand, and thrombopoietin. These results further suggest that LIF does not have a direct effect on expansion of human fetal CD34+ thy-1+ cells even at concentrations as
high as 10 ng/mL. Third, to further rule out a direct role of LIF in
facilitating ex vivo stem cell expansion, control studies with
neutralizing antibody against LIF were conducted. Our data demonstrated
that the production of murine LIF is up-regulated by the stromal cells
in the presence of 10 ng/mL of either recombinant human or murine LIF
(data not shown). Although the concentration of murine LIF was not
detectable by ELISA (R&D Systems, Minneapolis, MN; sensitivity is 50 pg/mL) in either SCM or SCM-LIF, we were able to demonstrate a 3-fold increase in LIF expression in the LIF-treated AC6.21 cells by RT-PCR
(data not shown). SCM-LIF was prepared from stromal cultures treated
with 500 pg/mL recombinant human LIF (R&D Systems). The concentration
of residual human LIF in the SCM-LIF was then determined to be in the
range of 200-250 pg/mL by ELISA. CD34+ thy-1+
cells purified from human fetal BM were cultured on 200% SCM-LIF for 3 weeks in the absence or presence of 0.1 to 10 µg/mL of neutralizing antibody against human LIF (R&D Systems) as previously
described.2 We found similar results, including 100% of
wells (20 of 20) that were CD34+
thy-1+/positive and an average of 9% (9 2) of
CD34+ thy-1+ cells in each well, for all
cultures including positive control (200% SCM-LIF without
neutralizing antibody) (data not shown). This result shows that
ex vivo stem cell expansion was not affected by the addition of
various concentrations (0.1, 1, and 10 µg/mL) of neutralizing
antibody against human LIF to the cultures in both the frequency of
CD34+ thy-1+/positive wells and the percentage
of CD34+ thy-1+ cells in the wells. This result
demonstrates that neutralizing antibody against LIF cannot block the ex
vivo stem cell expansion facilitated by the SCEPF activity in the
SCM-LIF and suggests that LIF does not contribute directly to the SCEPF
activity in the SCM-LIF. Taken together, our results do not support the
notion that LIF might have a direct role in facilitating ex vivo
expansion of hematopoietic stem cells.
3. Whether the augmented production of LIF by cord blood (CB)
mononuclear cells may directly and/or indirectly underly the increased
in vitro and in vivo clonogenicity of CB, as compared to adult
peripheral blood mononuclear cells (PBMC). Drs Kurre and Burdach
have presented their data to demonstrate that there is a higher
production (2- to 3-fold) of LIF by the CB mononuclear cells than the
adult PBMC during stimulation from interferon , tumor necrosis
factor , interleukin-1, or lipopolysaccharide. Combined
activation with anti-CD3 and interleukin-2 leads to a more than 20-fold
increase in LIF production by CB mononuclear cells, compared to a
7-fold increase seen with adult PBMC. This effect is blunted
(4- and 5-fold increase, respectively) after monocyte (CD14) depletion,
suggesting that T-cell-monocyte interaction is required. It is
interesting to note the correlation of their data with several
previously published reports, suggesting that LIF might involve
expansion of more primitive progenitors with greater replicative
potential.3-5 But many lines of evidence do not
support this notion. First, many studies have revealed ontogeny-associated differences in a variety of functional attributes of stem-cell proliferation and differentiation in both in vivo and in
vitro settings.6-8 It has been recently demonstrated that long-term engrafting cells were approximately enriched in CB
CD34+ cells 8-fold more than in adult PBMC
CD34+ cells, and each CB long-term engrafting cell had an
approximately 15-fold higher multilineage proliferative
capacity.8 Thus these results suggest that an
intrinsic qualitative difference might be responsible for the
remarkable difference between these 2 sources of stem/progenitor
cells.7,8 Second, LIF is a pleiotropic cytokine with
distinct hematopoietic activities. In vivo treatment of mice with
recombinant murine LIF induces thrombocytosis and increases the number
of hematopoietic progenitor cells in spleen and bone
marrow.9 It has been further determined that in vivo treatment with LIF expands the number of committed progenitor cells and
BM-repopulating cells that accelerate short-term hematopoietic reconstitution without increasing radioprotection.10 These
data do not support a direct role for LIF as a single factor for
promoting expansion of hematopoietic stem cells in vivo. Third, it is
generally believed that production of many cytokines other than LIF
will be increased by CB mononuclear cells with various stimulation. To
prove the concept that the augmented production of LIF by CB mononuclear cells may directly and/or indirectly underly the increased in vitro and in vivo clonogenicity of CB, as compared to
adult PBMC, it will be required to establish the panel of cytokines whose production have been up-regulated and to rule in or rule out
the contribution of these cytokines including LIF for the activity to increase in vitro and in vivo clonogenicity of CB mononuclear cells.
Footnotes
Supported by National Cancer Institute grants NCI PPG CA
30206, NCI CA 33572, and NCI CA 71866.
Chu-Chih Shih
Stephen J. Forman
Division of Hematology/Bone Marrow Transplantation
City of
Hope National Medical Center
Duarte, CA
| |
References |
1.
Shih CC, Hu MC, Hu J, Medeiros J, Forman SJ.
Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells.
Blood.
1999;94:1623-1636.
2.
Shih CC, Hu MC, Hu J, et al.
A secreted and LIF-mediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells.
Blood.
2000;95:1957-1966[Abstract/Free Full Text].
3.
Broxmeyer HE, Hangoc G, Cooper S, Ribeiro RC, Graves V, Yoder M, Wagner J, Vadhan-Raj S, Benninger L, Ribinstein P, Broun ER.
Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults.
Proc Natl Acad Sci USA.
1992;89:4109-4113.
4.
Gabutti V, Timeus F, Ramenghi U, Crescenzio N, Marranca D, Miniero R, Cornaglia G, Bagnara GP.
Expansion of cord blood progenitors and use for hematopoietic reconstitution.
Stem Cells.
1993;11Suppl 2:105-112.
5.
Imamura M, Zhu X, Han M, Kobayashi M, Hashino S, Tanaka J, Kobarashi S, Kasai M, Asaka M.
In vitro expansion of murine hematopoietic progenitor cells by leukemia inhibitory factor, stem cell factor, and interleukin-1 beta.
Exp Hematol.
1996;24:1280-1288.
6.
Holyoake TL, Nicolini FE, Eaves CJ.
Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow.
Exp Hematol.
1999;27:1418-1427[Medline]
[Order article via Infotrieve].
7.
Wang JC, Doedens M, Dick JE.
Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay.
Blood.
1997;89:3919-3924[Abstract/Free Full Text].
8.
Leung W, Ramirez M, Civin CI.
Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood.
Biol Blood Marrow Transplant.
1999;5:69-76[Medline]
[Order article via Infotrieve].
9.
Estrov Z, Talpaz M, Wetzler M, Kurzrock R.
The modulatory hematopoietic activities of leukemia inhibitory factor.
Leuk Lymphoma.
1992;8:1-7[Medline]
[Order article via Infotrieve].
10.
Pruijt JF, Lindley IJ, Heemskerk DP, Willemze R, Fibbe WE.
Leukemia inhibitory factor induces in vivo expansion of bone marrow progenitor cells that accelerate hematopoietic reconstitution but do not enhance radioprotection in lethally irradiated mice.
Stem Cells.
1997;15:50-55[Abstract/Free Full Text].
