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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3644-3652
Thymic Repopulation by CD34+ Human Cord Blood Cells After
Expansion in Stroma-Free Culture
By
Bruno Verhasselt,
Tessa Kerre,
Evelien Naessens,
Dominique Vanhecke,
Magda De Smedt,
Bart Vandekerckhove, and
Jean Plum
From the Department of Clinical Chemistry, Microbiology and
Immunology, University of Ghent, University Hospital of Ghent, Ghent,
Belgium; and the Flanders Interuniversity Institute for Biotechnology,
Ghent, Belgium (Vlaams Interuniversitair Instituut voor Biotechnologie,
VIB).
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ABSTRACT |
Thymic repopulation by transplanted hematopoietic progenitor cells
(HPC) is likely to be important for long-term immune reconstitution and
for successful gene therapy of diseases affecting the T-cell lineage.
However, the T-cell progenitor potential of HPC, cultured in vitro for
cell number expansion and gene transfer remains largely unknown. Here,
we cultured highly purified human umbilical cord blood (CB)
CD34+CD38 or
CD34+CD38+ cells for up to 5 weeks in
stroma-free cultures supplemented with various combinations of the
cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2
ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated
thymus-repopulating ability of expanded cells in vitro and in vivo.
After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of
CD34+CD38 CB cells generated T cells and
natural killer cells in the thymus. Limiting dilution
experiments demonstrated increase in the number of T-cell progenitors
during culture. After 3 weeks of culture, gene marked
CD34+CD38 CB cells injected in the human
thymus fragment transplanted in severe combined immunodeficient (SCID)
mice (SCID-hu) generated thymocytes expressing the
retroviral encoded marker gene GFP in vivo. Thus, our results show that
the progeny of CD34+CD38 CB cells cultured
for extensive periods, harbor thymus-repopulating cells that retain
T-cell progenitor potential after expansion and gene transfer.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CLINICAL USE OF cord blood (CB)
mononuclear cells in transplantation has indicated that successful
engraftment depends on the number of nucleated cells injected per kg
body mass.1,2 Given the limited number of nucleated cells
that can be isolated from a CB donation, in vitro expansion of
mononuclear cells and hematopoietic precursor cells (HPC) might
overcome the current limitations for the use of CB for transplantation
of larger children and adults.2 For retroviral gene
transfer, cell cycle induction is beneficial, or even essential when
using nonlentiviral vectors.3 Several investigators have
reported extensive, growth factor-induced in vitro expansion of CB HPC.
Early acting cytokines such as stem cell factor (SCF), thrombopoietin
(TPO), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), IL-6,
erythropoietin (Epo), granulocyte colony-stimulating factor (G-CSF),
and others were used to expand the number of CB cells over 1 or more
weeks of culture.4-10 Besides total cell count, the number
of colony-forming cells (CFC) and long-term culture-initiating cells
(LTC-IC)5-8 also increased. Severe combined immunodeficient
(SCID) mice repopulating cells (SRC), present at low frequency in the
CD34+CD38Lin and the
CD34CD38Lin
fraction of CB HPC,7,11,12 could be expanded modestly in short-term culture7,12 and in 1 report even extensively (up to 70-fold during 9 to 10 weeks of culture).9 Most of our
knowledge of lymphoid precursor potential of expanded HPC is restricted to B-cell generation.7,13 Few data are available on T-cell precursor potential or the thymus-repopulating capacity of expanded HPC,4,10,14 as this cannot be assessed in suspension
culture or in the nonobese diabetic (NOD)-SCID
repopulation model.15 Thymic-dependent T-cell generation
continues in adults16 and may contribute to immune
reconstitution after HPC transplant.17,18 Gene therapy
aimed at curing diseases affecting the T-cell lineage can only succeed
if the genetically modified HPC can generate this
lineage.17,19,20 We recently described an in vitro model that allows the study of thymic-dependent T-cell generation in fetal
thymus organ culture (FTOC) from transduced CB HPC, cultured for 3 days
in stroma-free suspension culture.21
Here, we show that highly purified
CD34+Lin umbilical CB cells harbor in
both the CD38 and CD38+ fraction
thymus-repopulating cells, which could generate T cells and natural
killer (NK) cells in FTOC, even after several weeks of culture in
cytokine-supplemented medium without stromal support. The number of
T-cell progenitors present in the
CD34+CD38Lin fraction
increased during culture. After 3 weeks of culture, transduced
CD34+CD38 cells could repopulate SCID-hu
thymus, generating in vivo T cells, NK cells, and dendritic cells
expressing the green fluorescent protein (GFP) marker gene.
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MATERIALS AND METHODS |
Monoclonal antibodies (MoAbs) and flow cytometry.
Mouse anti-human MoAbs used were CD56 (Immunotech, Marseille, France),
anti-HLA-A3 (American Type Culture Collection [ATCC], Rockville,
MD), and as described previously.21-23
MoAbs were labeled with fluorescein isothiocyanate (FITC),
phycoerythrin (PE), or allophycocyanin (APC), or conjugated to biotin
(BIO). Biotinylated antibodies were revealed by streptavidin (SA)-tricolor (TC) (Caltag Laboratories, San Francisco, CA), SA-PE (Becton Dickinson Immunocytometry Systems, Mountain View, CA [BDIS]), or SA-APC (BDIS). MoAbs from ATCC were FITC- or BIO-conjugated in our
lab by standard methods. Flow cytometry and cell sorting was performed
as described previously.21,23
Mice.
Fetal day 15 thymic lobes were isolated as described
previously21 from C.B.-17 scid/scid (SCID) or
NOD-LtSz-scid/scid (NOD-SCID) mice, bred in our specific
pathogen-free breeding facility. For generation of SCID-hu mice, small
pieces of fresh human fetal liver and thymus were transplanted under
the SCID mouse kidney capsule as described previously.24
Human tissue was obtained following the guidelines of the Medical
Ethical Commission of the University Hospital of Ghent. Animals were
treated according to the guidelines of the Laboratory Animal Ethical
Commission of the University Hospital of Ghent.
Purification of CD34+ CB cells.
Umbilical CB was obtained from full-term, healthy newborns and used
within 24 hours after collection for isolation of mononuclear cells as
described previously.21 Briefly, the mononuclear cell fraction, containing on the average 11,250 CD34+ and 570 CD34+CD38 cells per 106
CD45+ cells, was cryopreserved in liquid N2
until use. After thawing, cells were stained with glycophorin-A, CD19,
and CD7 FITC for immunomagnetic depletion. Unlabeled cells were
recovered and stained with CD1 FITC, CD3 FITC, CD4 FITC, CD8 FITC, CD34
PE, and CD38 BIO. Cells that were CD34 PE++, lineage
markers (Lin) FITC, and either CD38
BIO/SA-TC+ or CD38 BIO/SA-TC (called
CD34+CD38+ or
CD34+CD38, respectively) were sorted on
a fluorescence-activated cell sorting (FACS) Vantage (BDIS) cell sorter
equipped with an argon-ion laser (488 nm). Sort gate for
CD38 cells was set not to exceed fluorescence
intensity of 99% of cells stained with isotypic control antibody.
After sorting, on the average 1,100 CD34+CD38+Lin cells and 425 CD34+CD38Lin were
obtained per 106 mononuclear cells recovered after thawing.
Sorted populations are collectively called
CD34+Lin cells. Sorted cells were
checked for purity, which was always at least 99.0%.
Cell culture.
All cultures were performed at 37°C in a humidified atmosphere
containing 7.0% (vol/vol) CO2 in air. The cells were
cultured in Iscove's modified Dulbecco's medium (IMDM), supplemented
with penicillin (100 IU/mL), streptomycin (100 µg/mL), and 10%
heat-inactivated fetal calf serum (complete IMDM; all products from
Life Technologies, Paisley, UK). Cytokine-supplemented medium was
refreshed at least twice a week. To avoid medium
exhaustion,8 cell density was not allowed to exceed 2 × 106 cells/mL.
Single harvest cultures.
Sorted CD34+Lin cells were resuspended
in complete IMDM supplemented with cytokines as indicated and seeded in
96-well round-bottom tissue culture plates (Falcon; Becton Dickinson
Labware, Franklin Lakes, NJ) at 2,500 to 7,500 cells in 150 µL medium
per well. In the second week of culture, cells were transferred to
24-well plates, after resuspension and incubation of the well in Cell Dissociation Buffer (Life Technologies) to remove adherent cells from
the well surface. For a third week of culture, the cells were
transferred to 6-well tissue culture plates or to 25-cm2
tissue culture flasks (Falcon). After the culture periods indicated, cells were harvested, counted, analyzed for CD34 and CD38 expression, and 90% of the cells were coincubated with 1 fetal thymic lobe to
start FTOC. Expansion was calculated by dividing the number of cells
obtained at the end of the culture period by the starting cell number.
For FTOC-limiting dilution experiments, 3 parallel wells containing
1,000 sorted CD34+CD38 cells each were
seeded in medium supplemented with cytokines, as indicated, and
cultured as described above. After 3, 10, and 17 days of culture, cells
from 1 well were harvested. Resuspended cells were distributed over
fetal thymic lobes: 1 lobe with 75% of the cells, 3 lobes with 5% of
the cells each, and 5 to 8 lobes with 1% of the cells each. The latter
lobes therefore received the progeny equivalent to that of 10 CD34+CD38 cells.
For SCID-hu thymus repopulation, 2,500 sorted
CD34+CD38 cells were gene marked after
48 hours of culture. To this end, half of the medium volume was
replaced with viral supernatant, supplemented with cytokines (to keep
final cytokine concentration unchanged) and cells were seeded in
96-well flat-bottom non-tissue culture-treated plates (Falcon), coated
with RetroNectin (Takara Biomedicals, Otsu Shiga, Japan) as instructed
by the supplier. The retrovirus used encoded the marker gene, EGFP
(Clontech, Palo Alto, CA), constructed and produced as described
previously.21 The batch used in this report was shown to be
free of replication competent retrovirus and contained after thawing 1 × 106 transforming units/mL (titrated on Jurkat T
cells [ATCC], data not shown). Cells were further cultured for up to
3 days. After this period, wells were resuspended and incubated in cell
dissociation buffer to remove adherent cells from the well surface.
Transduction efficiencies of the cells were assayed by flow cytometry
on a small sample, remaining cells were seeded in 24-well tissue
culture plates and culture was continued as described above. After the culture periods indicated, cells were harvested, counted, and 90% of
the cells were injected intrathymically as described below.
Sequential harvest cultures.
In a separate series of experiments, sorted
CD34+Lin cells were cultured for
extended periods in wells that were harvested weekly, each time leaving
one fourth of the cells in culture. To start the culture, freshly
sorted cells were resuspended in complete IMDM supplemented with
cytokines as indicated and seeded in 96-well round-bottom tissue
culture plates at 6,500 cells in 150 µL medium per well. At day 3 of
culture, cells were harvested by resuspension and incubation of the
wells in cell dissociation buffer to remove adherent cells from the
well surface. Each well was visually inspected to check for removal of
all adherent cells. One fourth of the harvested cells was reseeded in
the same well in cytokine-supplemented medium, while the remaining
cells were analyzed for CD34 and CD38 expression and in some
experiments, 90% of these cells were coincubated with 1 fetal thymic
lobe to start FTOC. This procedure was repeated every week of culture, reducing the cell number to one fourth every week, for 6 weeks. In this
way, at day 3 of culture, one fourth of the progeny of original cell
input is reseeded. These cells are obtained at the second harvest (day
10 of culture) and at that moment, therefore, constitute of one fourth
of the progeny of the original cell input. Again, one fourth of this
progeny is reseeded and harvested 1 week later. Consequently, after 17 days of culture (third harvest), the harvested cells constitute 1/16th,
and after 38 days of culture (sixth harvest), 1/1,024th of the progeny
of original cell input. This means that without expansion, a well
initiated with 6,500 cells would contain after 38 days of culture about
6 input cells. Expansion was calculated by multiplying the number of
cells counted on harvest with 4n, where n is the number of
harvests preceding, and dividing this product by the starting cell number.
FTOC.
Hanging drops were prepared in Terasaki plates by adding per well 25 µL complete IMDM containing the progeny or a fraction thereof of
CD34+Lin cells after washing in complete
cytokine-free medium. To each well, 1 fetal thymic lobe was added, the
plates were inverted to form hanging drops and incubated during 72 hours. After this incubation, at day 0 of FTOC, the lobes were removed,
washed in complete medium, and cultured for 30 days as described
previously.21 For limiting dilution experiments, harvested
cells were coincubated with NOD-SCID lobes and irradiated
CD34+ cells as described previously.25
Cytokines.
All cytokines were used at a fixed concentration during culture: 100 ng/mL recombinant human SCF (R&D Systems Europe, Abingdon, UK), 100 ng/mL recombinant human FL (R&D Systems Europe), 20 ng/mL TPO (R&D
Systems Europe), 10 ng/mL recombinant human IL-3 (Innogenetics, Antwerp, Belgium), 100 U/mL recombinant human IL-6 (produced in yeast,
kindly provided by Dr W. Fiers, University of Ghent, Belgium; specific
activity was 280 U/ng, assayed as described previously26).
SCID-hu thymic repopulation.
SCID-hu mice, transplanted 5 to 20 months before, were given a
sublethal dose of whole-body irradiation (200 cGy, 40 cGy/min) using a
cobalt irradiation device. The next day, they were anesthetized and the
kidney carrying the human tissue was dissected free. Donor cells were
washed extensively, resuspended in 20 µL of complete cytokine-free
medium, and injected intrathymically. Thymic implants were removed and
analyzed by flow cytometry, 30 days postinjection. HLA mismatch between
CB donors (HLA-A3+) and thymus acceptors
(HLA-A3) allowed differentiation between acceptor
thymocytes and total donor or transduced donor-derived thymocytes by
gating on donor HLA haplotype or on GFP expression, respectively.
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RESULTS |
Expansion of human CD34+Lin CB
cells in stroma-free liquid cultures.
Before analyzing thymic repopulation by cultured CD34+ CB
cells, we first compared efficacy of expansion in 4 different cytokine mixes: TPO + FL ± SCF and IL-3 + SCF ± IL-6. Highly purified
CD34+CD38+ and
CD34+CD38 CB cells were cultured in
medium supplemented with these cytokine mixes. After 17 days of
culture, the total progeny was harvested (single harvest culture).
Total and especially CD34+ cell number expansion was higher
in cultures started with CD34+CD38 cells
compared with CD34+CD38+ cells
(Fig 1). Expansion in TPO + FL-supplemented
cultures was inferior to the other combinations tested. In cultures
initiated with CD34+CD38+ or
CD34+CD38 CB cells, supplemented with
TPO + FL, CD34+ cell number expanded on the average 4.1- and 7.2-fold, respectively (resp). Addition of SCF to
this mixture increased CD34+ cell number expansion to 6.1 resp 26.6-fold and total cell number expansion was
5-fold higher. Cultures supplemented with IL-3 + SCF and IL-3 + SCF + IL-6 showed comparable total and CD34+ cell number
expansion as TPO + FL + SCF-supplemented cultures. Almost no viable
cells were recovered from cells cultured in complete medium without
cytokines (data not shown). To assay
CD34+CD38 CB cell expansion for
extensive culture periods in manageable culture dimensions, we
performed a series of experiments in which total cell number was weekly
reduced to a fourth (sequential harvest cultures). Cell number
expansion was calculated as described in Materials and Methods. Total
cell numbers expanded continuously over the culture period of 38 days.
After 17 days of culture, total (Fig 2A)
and CD34+ (Fig 2B) calculated cell number expansion was
very similar to the values measured in single harvest culture (Fig 1).
Again, TPO + FL was inferior in inducing total and CD34+
cell number expansion compared with the other cytokine combinations. Moreover, the CD34+ cell content declined after more than 3 weeks of culture in TPO + FL-supplemented cultures. In the other
combinations tested, CD34+ cell content increased during at
least 30 days, exceeding a 100-fold expansion. As reported
before,13 most of the progeny of
CD34+CD38 cells express CD38 after more
than 1 week of culture. However, a minority retained the
CD38 phenotype over extended culture periods, most
prominent in cultures supplemented with TPO + FL + SCF
(Fig 3A and data not shown).
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| Fig 1.
Expansion of total and CD34+ cell number in
single harvest cultures. Sorted
CD34+CD38+Lin or
CD34+CD38Lin cells were
cultured for 17 days in medium supplemented with cytokines as
indicated. Columns represent average expansion (fold increase of start
cell number) from independent experiments with 6 different donors,
error bars indicate standard deviation (SD). Dotted columns, all cells;
filled columns, CD34+ cells.
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| Fig 2.
Expansion of total and CD34+ cell number in
sequential harvest cultures. Sorted
CD34+CD38Lin cells were
cultured in medium supplemented with cytokines and harvested after
different culture periods as indicated. Points represent average
expansion (fold increase of start cell number) of all cells (A) or
CD34+ cells (B) from independent experiments with 3 different donors, error bars indicate SD. ( ) TPO + FL, ( ) TPO + FL + SCF, ( ) IL-3 + SCF, ( ) IL-3 + SCF + IL-6.
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| Fig 3.
Thymic repopulation by cultured
CD34+CD38Lin cells in
vitro. (A) Flow cytometric analysis of the progeny of
CD34+CD38Lin cells after
pre-FTOC culture for 10, 24, and 38 days in TPO + FL + SCF. Dot
plots show CD38 FITC versus CD34 PE staining. (B) Flow cytometric
analysis of thymocytes recovered from FTOC initiated with cells shown
above in (A). Dot plots show CD4 APC versus CD8 PE, anti-TCR- 
FITC versus CD3 APC and CD56 PE versus CD3 APC stainings. The latter
staining was not performed on thymocytes from FTOC initiated with
CD34+CD38Lin cells cultured
for 10 days. Quadrants were set to include 99% of cells stained with
isotypic control antibody in lower left quadrants. Data shown are
representative of 2 independent experiments with 2 different donors.
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CD34+Lin CB cells retain the
potential to generate thymocytes in vitro after extensive culture.
To investigate whether the lymphoid precursor potential is retained in
cultured CD34+Lin CB cells,
CD34+CD38+ and
CD34+CD38 CB cells grown in single
harvest cultures for 3, 10, and 17 days were assayed in FTOC. The
results are summarized in Table 1. As
described before,21 after 30 days of FTOC, thymocytes
expressing CD4, CD8, and CD3 were present if thymic repopulation
succeeded (more than 80% of FTOCs performed with freshly sorted
CD34+Lin CB cells). FTOC phenotype after
successful repopulation (scored by the presence of
CD4+CD8+ double-positive (DP) immature
thymocytes and/or CD3+T-cell receptor
[TCR]- thymocytes) was comparable to that
obtained with CD34+Lin CB cells grown in
sequential harvest cultures (vide infra and Fig 3B). Both
CD34+CD38+ and
CD34+CD38 cells of almost all CB donors,
grown for 3 days in any of the cytokine mixes tested, could generate
CD3+ T cells and DP thymocytes in FTOC (Table 1).
CD34+CD38+ cultured for 1 or 2 additional weeks
showed a somewhat reduced T-cell generation success rate. Some FTOCs
then contained only TCR-+ cells and NK cells, but no
DP or CD3+TCR- thymocytes. This
FTOC phenotype was not considered as thymic repopulation, as
TCR-+ cells and NK cells can develop
extrathymically.27 By contrast, with all CB donors,
CD34+CD38 cells cultured for 17 days
generated CD3+TCR- T cells and
frequently also DP thymocytes in FTOC, irrespective of the cytokine mix
used in the pre-FTOC culture (Table 1).
CD3CD56+ NK cells were present in most
FTOCs (data not shown). These data indicate that the progeny of both
the CD38+ and CD38 subsets of
CD34+Lin CB cells contained cells with
T-cell and/or NK cell progenitor potential, even after 17 days of
culture in the presence of cytokines.
By using the cells obtained during sequential harvest cultures as
described above, we could address how long we could extend stroma-free
culture while maintaining T-cell and NK cell progenitor potential. Due
to previous harvests, a decreasing fraction of the progeny of original
cell input is obtained as described in Materials and Methods. Quiescent
cells are therefore diluted out. For 2 CB donors, we analyzed in vitro
thymocyte generation from CD34+CD38+ and
CD34+CD38 cells obtained weekly in
sequential harvest cultures over more than 5 weeks. None of these 2 donors generated DP thymocytes in FTOC from
CD34+CD38+ cells cultured for more than 17 days, irrespective of the cytokine mix used. The progeny of
CD34+CD38 cells, cultured for a maximum
of 24 days in medium supplemented with TPO + FL or IL-3 + SCF, could
repopulate thymi (data not shown). By contrast, CD3+ T
cells and DP thymocytes were generated in FTOC started with CD34+CD38 cells obtained after up to 38 days of sequential harvest culture in medium supplemented with TPO + FL + SCF or IL-3 + SCF + IL-6. As an example, Fig 3B shows the phenotype
of FTOC initiated with CD34+CD38 cells
from the same donor cultured for 10, 24, or 38 days in medium
supplemented with TPO + FL + SCF. Both
CD3+TCR+ and
CD3+TCR T cells and
CD3CD56+ NK cells were generated in
these FTOCs (Fig 3B). T-cell development was still continuing at the
end of FTOC, indicated by the presence of a population of DP
thymocytes. With increasing pre-FTOC culture periods, the fraction of
DP thymocytes generated by cultured
CD34+Lin cells decreased (Fig 3B and
data not shown). As described in Materials and Methods, without
expansion, wells harvested to start FTOC after 38 days of sequential
harvest culture would contain about 6 sorted
CD34+CD38 input cells. However, due to
expansion, in TPO + FL + SCF resp IL-3 + SCF + IL-6-supplemented cultures, wells contained on the average of 90 × 103 resp 42 × 103
cells, including 3.2 × 103 resp 5.9 × 103 CD34+ cells.
Expansion of thymus-repopulating cells in cultures of
CD34+CD38Lin
CB cells.
In single harvest cultures described above, almost the total progeny is
put in FTOC. Quiescent cells present at the start of culture could in
theory be ultimately harvested to initiate FTOC. However, T-cell
generation from cultured CD34+CD38 cells
obtained during sequential harvest culture seemed to indicate that the
number of putative T-cell progenitors increased during culture. To
demonstrate actual expansion of putative T-cell progenitors, we
performed limiting dilution experiments as described in Materials and
Methods with 3 different CB donors. Table 2
shows representative results obtained with 1 of these donors. After 3 days of culture, the progeny equivalent to that of 750 CD34+CD38 cells could repopulate the
thymic lobe, irrespective of the cytokine mix where these cells were
cultured in (data not shown). However, the progeny equivalent to that
of 50 or 10 cells could not in almost all FTOCs performed (Table 2).
The cell number recovered after 3 days of culture of
CD34+CD38 cells is about the same as the
input number, as we previously described.21 After 10 days
of pre-FTOC culture, the repopulated fraction in the lobes that
received the progeny of 50 CD34+CD38
cells cultured in TPO + FL + SCF and IL-3 + SCF ± IL-6 increased (Table 2). FTOCs initiated with the progeny equivalent to that of 10 cells were only repopulated by cells cultured in medium supplemented
with TPO + FL + SCF. An additional week of culture to a total of 17 days in this cytokine mix did not further increase repopulation
frequency (Table 2). After 17 days of culture in IL-3 + SCF ± IL-6-supplemented medium, the progeny equivalent to that of 10 cells
could now also repopulate thymic lobes. The progeny equivalent to that
of 10 or 50 CD34+CD38 cells cultured in
TPO + FL could not generate DP or
CD3+TCR- + thymocytes in almost all FTOCs
performed. Overall, these experiments showed an increase in the number
of thymus-repopulating cells in the progeny of
CD34+CD38 cells, when cultured in TPO + FL + SCF or in IL-3 + SCF ± IL-6-supplemented medium.
In vivo thymic repopulation by gene marked CD34+
CD38Lin CB cells after
extensive culture.
The experiments we performed indicated that after extended suspension
culture a progeny equivalent to that derived from a few
CD34+CD38 cells could generate
thymocytes. Limiting dilution experiments indicated that the number of
putative thymus-repopulating cells increased during culture. To prove
that these cells divided in vitro and can generate thymocytes after
gene transfer, we transduced CD34+CD38
cells cultured in TPO + FL + SCF or IL-3 + SCF + IL-6-supplemented medium with a Moloney murine leukemia virus-derived retrovirus encoding
the marker gene GFP. One day after transduction, the percentage of
cells expressing the retroviral encoded GFP was on the average (N = 3)
36%. Gene transfer requires division so that GFP+ cells
are the progeny of cells that were replicating at the moment of
transduction.21 We previously showed that after 3 days of culture in IL-3 + SCF-supplemented medium, transduced
CD34+CD38 cells generate thymocytes
expressing the marker gene in FTOC.21 In the present study,
because culture of CB cells to increase graft size and gene transfer
will probably last longer, we wanted to determine whether transduced
CD34+CD38 cells, cultured for longer
than 3 days, can repopulate thymus in vivo. Therefore, we performed a
series of SCID-hu thymus repopulation23,28 experiments with
almost the complete progeny of retrovirally transduced CD34+CD38 cells, cultured in medium
supplemented with either TPO + FL + SCF or IL-3 + SCF + IL-6,
and obtained with both cytokine mixes similar results.
Figure 4 shows the phenotype of the human
thymus, 1 month after intrathymic injection of the progeny of
CD34+CD38 cells, cultured for 3 weeks in
TPO + FL + SCF. In this example (Fig 4A), more than 75% of the SCID-hu
thymocytes were from CB donor origin (HLA-A3+), of which
about 1 in 4 expressed the marker GFP (Fig 4B). Acceptor thymocytes
(HLA-A3) were mainly mature, radioresistant
CD3+, CD4+CD8
single-positive (SP) or CD4CD8+ SP
thymocytes (Fig 4C). Donor-derived thymocytes were predominantly of the
immature DP phenotype, with variable CD3 cell surface expression levels. As observed previously in vitro,21 GFP-expressing
thymocytes seem to develop somewhat slower compared with nontransduced
donor thymocytes, as they generated less SP thymocytes 30 days after intrathymic injection. Lineage distribution in gene marked thymocytes was similar to that of other donor-derived thymocytes: a minority of
the CD3hi cells were TCR+, and small
populations of GFP+CD3CD56+
NK cells, HLA-DRhiCD4+ dendritic cells and
CD34+ precursor cells were present (data not shown).
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| Fig 4.
Thymic repopulation by cultured
CD34+CD38Lin cells in vivo.
Flow cytometric analysis of SCID-hu thymocytes, 30 days after injection
with transduced
CD34+CD38Lin cells,
precultured for 3 weeks in TPO + FL + SCF. (A) Dot plot shows
anti-HLA-A3 FITC staining plus GFP fluorescence versus CD3 APC
staining. (B) Dot plot shows GFP fluorescence versus CD3 APC staining;
cells were not stained with anti-HLA-A3 FITC. (C) Dot plots show CD4
APC versus CD8 and CD4 APC versus CD3 PE staining for cells stained
with anti-HLA-A3 FITC as in (A), gated on either FITC
GFP cells (acceptor cells) or FITC+
GFP+ cells (donor cells); and for cells not stained with
anti-HLA-A3 FITC as in (B), gated on GFP+ cells
(transduced GFP+ donor cells). Quadrants were set to
include 99% of cells stained with isotypic control and
GFP cells in lower left quadrants. Data shown are
representative of 2 independent experiments with 2 different donors.
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DISCUSSION |
In this report, we investigated thymus-repopulating potential of
expanded CD34+Lin CB cells. Our data
indicate that expanded CD34+Lin CB cells
contain thymus-repopulating cells in both the CD38
and CD38+ fraction, even after several weeks in stroma-free
culture. After 38 days of culture, the progeny of
CD34+CD38 cells could generate
thymocytes in vitro. During culture, the number of thymus-repopulating
cells increased. After 3 weeks of culture, transduced
CD34+CD38 cells could repopulate SCID-hu
thymus, generating in vivo T cells, NK cells, and dendritic cells
expressing the GFP marker gene. Collectively, our data indicate that
thymus-repopulating cells can be expanded in vitro and can generate
transduced thymocytes in vivo after extensive culture and retroviral
gene transfer in vitro.
Clinical experience with the use of CB cells as a transplantable source
of HPC indicates that myeloid and platelet engraftment is delayed if
fewer than 20 to 30 million nucleated CB cells per kg body mass are
injected.1,2 As the median number of nucleated cells in CB
donations is about 1,000 million or less, expansion of CB cells might
be necessary to use these widely accessible HPC in transplantation of
adults.2 Therefore, considerable amount of data has been
produced on growth factor-induced proliferation of primitive,
hematopoietic CB cells in vitro.4-10 We compared 2 types of
cytokine mixes known to act on primitive hematopoietic cells: TPO + FL ± SCF and IL-3 + SCF ± IL-6. TPO was recently described to act
as a growth factor for CD34+ HPC of different origins,
including CB.6,9,29,30 Combination of TPO with FL, the
ligand for the flt3/flk-2 receptor tyrosine kinase, was reported by
Piacibello et al6 to induce massive expansion of
CD34+ and CD34+CD38 CB cells
in sequentially harvested cultures. In their study, CD34+
cells cultured in medium supplemented with TPO + FL expanded more than
1,000-fold in 3 to 5 weeks of culture. After 25 weeks of culture,
CD34+CD38 cell numbers were calculated
to have increased more than 10 million-fold.6 Recently, the
same group reported extensive SRC expansion in TPO + FL-supplemented
cultures of CD34+CB cells.9 In our experiments,
TPO + FL was not able to sustain extensive expansion of sorted
CD34+CD38 CB cells. In fact, addition of
SCF increased total cell number expansion and was essential to maintain
CD34+ cells in sequentially harvested cultures lasting
longer than 3 weeks (Fig 2). Moreover, addition of SCF was necessary to
expand thymus-repopulating cells (Table 2). This is in line with
previous reports that addition of SCF to TPO + FL increases
several-fold the expansion of CD34+ bone marrow cells after
6 to 14 days of culture.30,31 TPO or FL as single factors
could support LTC-IC expansion from
CD34+CD38 bone marrow cells cultured in
serum-free medium for 10 days.29 However, this was not the
case for TPO in CB LTC-IC expansion in serum-free culture for 10 days.8 In the latter study, addition of SCF was shown to be
important for CB CFC expansion.8 We do not have an
explanation why we could not reproduce the observations of Piacibello
et al.6 These investigators have used cytokines from a
different source and in slightly different concentrations than we did.
In both their and our experiments, culture medium contains 10%
(vol/vol) fetal calf serum. FCS is known to contain factors affecting
human HPC proliferation and maintenance in vitro.32 Serum-free culture media have been successfully used for expansion of
CD34+ CB cells.7,8 Use of these media will
eliminate the variability and potential biohazards introduced by FCS.
As others and we observed,8 cultures of CD34+
CB cells contain a portion of adherent cells. Transfer of cells between
the adherent and the suspension phase could bias calculations in
sequentially harvested cultures. We detached all adherent cells at each
harvest. At the third harvest (17 days of culture), cell number
expansion calculated from sequentially harvested cultures was
comparable to that measured in single harvest cultures (compare Fig 2
with Fig 1). Similarly, this bias was also excluded in the recent study
of Piacibello et al.9
A classical cytokine cocktail frequently used to expand HPC is IL-3 + SCF + IL-6.4,11,33,34 We compared this mixture with the
combination IL-3 + SCF, previously shown to maintain in
vitro21 and in vivo28 thymus-repopulating
ability of gene-marked CD34+ CB cells cultured for 3 to 4 days. Our experiments did not show a difference between cultures
supplemented with either of these cocktails in expansion of total;
CD34+, or thymus-repopulating cell number as estimated by
the limiting dilution-FTOC experiments. Interestingly,
CD34+CD38 cells cultured in IL-3 + SCF-supplemented medium for 7 days, but not freshly sorted
CD34+CD38 cells, produced IL-6
bioactivity in medium conditioned for 4 days (data not shown).
Therefore, endogenous IL-6 production in IL-3 + SCF-supplemented
cultures may blur the difference induced by addition of exogenous IL-6.
However, thymic repopulation by CD34+CD38 cells seemed to be maintained
longer after culture when exogenous IL-6 was added to IL-3 + SCF.
Presence of IL-6 at the start of the culture may be important. In
single harvest cultures of CD34+CD38 CB
cells lasting 10 days, multivariate analysis of cytokine combinations by Zandstra et al8 showed that IL-6 is important in CB
LTC-IC and CFC expansion. In our experiments, the mix IL-3 + SCF + IL-6 seemed equivalent to TPO + FL + SCF in cell number expansion and thymic
repopulation by expanded CD34+CD38 CB
cells. However, the number of thymus-repopulating cells seemed to
increase more prominently in TPO + FL + SCF compared with IL-3 + SCF + IL-6-supplemented cultures. In addition, similar results were obtained
with single harvest cultures supplemented with all 5 cytokines (data
not shown).
The progeny of CD34+CD38+ cells showed a
reduced potential in generating T cells in vitro compared with that of
CD34+CD38 cells with increasing culture
duration, regardless of the cytokine mix used. This suggests that both
the CD34+CD38+ and the
CD34+CD38 population of CB cells contain
thymus-repopulating cells, analogous to myeloid CFC. However,
thymus-repopulating cells are lost after extended culture of
CD34+CD38+ cells. By contrast, after more than
5 weeks in culture, thymus-repopulating cells were found in 1/1,024th
of the progeny of 6,500 CD34+CD38Lin cells.
Recently, the frequency of thymus-repopulating cells was estimated to
be between 1/100 and 1/500 in freshly isolated CD34+ CB
cells.25 The limiting dilution experiments and gene
transfer experiments we performed show that the progeny of
CD34+CD38 cells contains expandable
thymus-repopulating cells. Such cells would represent long-term culture
thymus-repopulating cells, similar to myeloid LTC-IC. After 38 days of
culture, the progeny of CD34+CD38 cells
cultured in medium supplemented with TPO + FL + SCF or IL-3 + SCF + IL-6 contained mainly myeloid lineage marker CD33+ cells,
for the majority monocytes (CD14+HLA-DR+), but
no T cells, NK cells, or B cells (data not shown). This indicates that
the T cells and NK cells recovered from the FTOC were generated in the
thymus from precursor cells. It is unclear whether these precursor
cells might represent the human equivalent of a common lymphocyte
progenitor (CLP), recently identified in mice.35 Our data
indicate that thymus-repopulating CB T-cell and/or NK cell progenitors
can be kept in culture in a stroma-free environment for more than 5 weeks. Previous work by Galy et al14 demonstrated SCID-hu
thymus repopulation by CD34+ bone marrow cells, cultured
for 3 weeks on autologous bone marrow stroma. Partially purified CB
CD34+ cells were shown to repopulate SCID-hu thymus after 1 week of culture in IL-3 + SCF + IL-6-supplemented medium.4
Also, lymphoid marker CD2-expressing cells could be found in TPO + FL-supplemented cultures of CD34+ CB cells, after 12 weeks
of culture. However, this study did not clarify whether these cells
were NK cells or possibly T cells.6
As we show here, transgenic thymocytes can be generated in SCID-hu
thymus from transduced CD34+CD38 CB
cells expanded during 3 weeks of stroma-free culture. This period is
sufficient for reaching graft sizes that would contain enough nucleated
cells and HPC to engraft adults. Retroviral marking in our experiments
showed that thymocytes could be generated by cells that divided around
day 2 of culture. We injected the cells directly into the human thymus,
as thymic homing in SCID-hu mice is inefficient and not a validated
model for human thymic seeding.36 Therefore, we cannot
exclude that the thymus-repopulating cells we describe here might fail
to home to the thymus after intravenous injection in humans.
The thymus remains functional and generates naive T cells in
adults.16 In acquired immunodeficiency syndrome (AIDS)
patients, thymic T-cell generation is responsible for the increase in
naive T-cell counts during highly active antiretroviral
therapy.16 Gene therapy for diseases affecting T cells,
such as adenosine deaminase (ADA) deficiency, AIDS,
and X-linked SCID, requires persistent lymphoid precursor potential of
the genetically modified precursors.19,20,37 As our data
show that the progeny of transduced CD34+CD38 cells can repopulate the
thymus in vivo after 3 weeks of culture, thymic repopulation by
transplanted CB cells after expansion and gene transfer may be
possible, contributing to immune reconstitution and success of gene therapy.
| |
ACKNOWLEDGMENT |
We thank Christian De Boever for artwork, Achiel Moerman, Veronique
Debacker, Isabelle Windey, Ilse Swennen, and Greet De Smet for animal
care, the Departments of Obstetrics, Cardiac Surgery, and Pathology for
the supply of human tissue, the Department of Radiotherapy and Nuclear
Medicine for irradiation facilities, Dr Jo van Damme for measuring IL-6
bioactivity, and Dr G. Leclercq for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted April 1, 1999; accepted July 29, 1999.
Supported by grants from the Gezamelijk Overlegde Actie (GOA)
University of Ghent; the Fund for Scientific Research, Flanders (Belgium); and the VIB. B.V. and T.K. are research assistants and D.V.
is a postdoctoral research fellow of the Fund for Scientific Research,
Flanders (Belgium). E.N. is a VIB employee.
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.
Address reprint requests to Bruno Verhasselt, MD, Department of
Clinical Chemistry, Microbiology and Immunology, University of Ghent,
University Hospital of Ghent, 4 Blok A De Pintelaan 185, B-9000 Ghent,
Belgium; e-mail: Bruno.Verhasselt{at}rug.ac.be.
| |
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ABSTRACT
INTRODUCTION