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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3269-3270
CORRESPONDENCE
Quality of Repopulation in Nonobese Diabetic Severe Combined
Immunodeficient Mice Engrafted With Expanded Cord Blood
CD34+ Cells
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LETTER |
To the Editor:
In a recent issue of Blood, Guenechea et al1
reported that ex vivo cytokine-expanded CD34+ cord blood
cells retained nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating activity of fresh cells, although there was a
significant delay in the time to achieve similar levels of engraftment.
They also found that there was little change in the quality of
engraftment in terms of represented lineages 120 days after
inoculation, although they suggest a trend toward a reduction of the
myeloid (CD33+) compartment.
To develop a clinically applicable protocol, we have recently examined
the potential for expansion of cord blood progenitors under serum-free
conditions. As part of this study, we analyzed the characteristics of
NOD/SCID engraftment of cells cultured ex vivo for 14 days (Fig
1). In contrast to the findings reported above, we found that culture with interleukin-6 (IL-6) (10 ng/mL), IL-11 (10 ng/mL), Flt3-ligand (FL) (50 ng/mL), and
thrombopoietin (TPO) (10 ng/mL) was associated with a switch from
dominant B lymphopoiesis (CD19+) toward myelopoiesis
(CD13+) in animals analyzed after 40 days in the absence
of in vivo cytokine support. Furthermore, in other studies using
alternative combinations of cytokines (IL-3 [20 ng/mL], IL-6 [20
ng/mL], stem cell factor [SCF] [100 ng/mL], and FL [100 ng/mL]),
we found that the same switch in engraftment quality occurred after 48 hours of ex vivo culture (Demaison et al, in press). These
findings can be interpreted in two ways. Firstly, this may reflect a
forced commitment of the mutipotent SCID-repopulating cell (SRC) to
myelopoiesis, and/or selective inhibition of B-lymphoid commitment, and
is similar phenotypically to the patterns of engraftment observed in
NOD/SCID mice supplemented in vivo with FL either alone or in
combination with IL-7 and SCF, or a combination of SCF,
granulocyte-macrophage colony-stimulating factor (GM-CSF), and
IL-3.2 If this is the case, our findings suggest that these
changes in commitment are initiated by exposure to cytokines in vitro
and, more importantly, do not revert spontaneously in vivo. Secondly,
there may be a selective expansion or retention of SRCs intrinsically
restricted to the myeloid lineage. This possibility is supported by
previous studies that used retroviral marking or radiation-induced
chromosomal aberrations to track distinct cell lineages in mice, and
our own recent experiments that indicate that murine retroviral vectors can selectively mark ex vivo-cultured human CD34+ cells
that repopulate NOD/SCID mice in a myeloid-restricted
pattern3,4 (Demaison et al, in press).
Furthermore, good evidence has emerged for a murine clonogenic common
lymphoid precursor (CLP) population that possesses lymphoid-restricted
(T, B, and natural killer) repopulating activity.5
Heterogeneity within the SRC compartment is not surprising. For
example, a small population of CD34 lin
cells has the capacity to repopulate in a pattern similar to that of
cord blood and adult CD34+ populations.6
Conversely, second-gestation human fetal blood samples engraft in the
absence of cytokine support with a pronounced erythroid bias when
compared with cord blood or adult samples (Pahal G and
Thrasher AJ, unpublished observations, 1999).
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| Fig 1.
Lineage distribution in NOD/SCID mice engrafted with
fresh cord blood CD34+ cells or cord blood
CD34+ cells cultured for 14 days in serum-free medium
supplemented with IL-6 (10 ng/mL), IL-11 (10 ng/mL), FL (50 ng/mL), and
TPO (10 ng/mL). Nine mice engrafted with 2 × 105 to
106 CD34+ cells from 2 cord samples were
analyzed in each group.
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As clearly shown in another recent publication, the time for
reconstitution after cord blood transplantation, which is directly related to the cell dose, may be a significant limiting
factor.7 Selective expansion of alternative repopulating
cell populations, such as those restricted to the myeloid lineage, may
therefore be of significant benefit for hematopoietic support in the
early time period after cord transplantation. At the same time,
potential changes in the commitment of repopulating cells initiated by
ex vivo culture need to be clarified. The reasons for the differences in engraftment quality noted in Guenechea's study and our own are not
entirely clear but could relate to the timing of analysis (120 v 40 days), or to differences in ex vivo culture conditions. Further studies on the quantitation, kinetics, and quality of repopulating cell engraftment after ex vivo culture of cord blood cells
are therefore warranted if stem cell expansion protocols are to be
effectively optimized for clinical use.
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ACKNOWLEDGMENT |
A.J.T. is a Wellcome Clinical Scientist.
Michael P. Blundell
Christophe Demaison
Gaby Brouns
Jacki P. Goldman
Hubert B. Gaspar
Christine Kinnon
Adrian J. Thrasher
Molecular Immunology Unit
Institute of Child Health
London,
UK
Lorenza Lazzari
Girolamo Sirchia
Milano Cord Blood
Bank
Centro Trasfusionale e di Immunologia dei Trapianti
IRCCS
Milano, Italy
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REFERENCES |
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Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34+ cord blood cells.
Blood
93:1097, 1999[Abstract/Free Full Text]
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Kapp U, Bhatia M, Bonnet D, Murdoch B, Dick JE:
Treatment of non-obese diabetic (NOD)/severe-combined immunodeficient mice (SCID) with flt3 ligand and interleukin-7 impairs the B-lineage commitment of repopulating cells after transplantation of human hematopoietic cells.
Blood
92:2024, 1998[Abstract/Free Full Text]
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Abramson S, Miller RG, Phillips RA:
The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems.
J Exp Med
145:1567, 1977[Abstract/Free Full Text]
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Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice.
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Identification of clonogenic common lymphoid progenitors in mouse bone marrow.
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Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE:
A newly discovered class of human hematopoietic cells with SCID-repopulating activity.
Nat Med
4:1038, 1998[Medline]
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Rubinstein P, Carrier C, Scaradavou A, Kurtzberg J, Adamson J, Migliaccio AR, Berkowitz RL, Cabbad M, Dobrila NL, Taylor PE, Rosenfield RE, Stevens CE:
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