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HEMATOPOIESIS
From the Stem Cell Institute, the Department of
Medicine and Cancer Center, University of Minnesota, Minneapolis; the
Department of Medicine, the Department of Veterans Affairs Medical
Center, University of Nevada, Reno; and the Department of Molecular
Biology, Princeton University, Princeton, NJ.
This report describes stroma-based and stroma-free cultures that
maintain long-term engrafting hematopoietic cells for at least 14 days
ex vivo. Umbilical cord blood (UCB) CD34+ cells were
cultured in transwells above AFT024 feeders with
fetal-liver-tyrosine-kinase (FL) + stem cell factor (SCF) + interleukin 7 (IL-7), or FL + thrombopoietin (Tpo).
CD34+ progeny were transplanted into nonobese
diabetic-severe combined immunodeficiency (NOD-SCID) mice or preimmune
fetal sheep. SCID repopulating cells (SRC) with multilineage
differentiation potential were maintained in FL-SCF-IL-7 or FL-Tpo
containing cultures for up to 28 days. Marrow from mice highly
engrafted with uncultured or expanded cells induced multilineage human
hematopoiesis in 50% of secondary but not tertiary recipients. Day 7 expanded cells engrafted primary, secondary, and tertiary fetal sheep.
Day 14 expanded cells, although engrafting primary and to a lesser
degree secondary fetal sheep, failed to engraft tertiary recipients. SRC that can be transferred to secondary recipients were maintained for
at least 14 days in medium containing glycosaminoglycans and cytokines
found in stromal supernatants. This is the first demonstration that ex
vivo culture in stroma-noncontact and stroma-free cultures maintains
"long-term" engrafting cells, defined by their capacity to engraft
secondary or tertiary hosts.
(Blood. 2001;97:3441-3449) Ex vivo expansion of hematopoietic cells is needed
not only for gene transfer1-3 and tumor
purging,4,5 but also to amplify the number of
hematopoietic stem cells (HSC), especially in umbilical cord blood
(UCB) grafts. The use of UCB as a source of HSC is increasing because
there is less incidence of graft-versus-host disease after UCB
transplantation.6,7 This allows use of grafts with greater
HLA disparity and therefore increases the number of grafts available
for transplantation. However, absolute HSC dose is a limiting factor in
the use of UCB as a graft for adult transplant recipients.
Hematopoiesis is a tightly regulated process in which HSC give rise to
all components of the hematopoietic system for the lifetime of the
individual. Properties of HSC are self-renewal, multilineage
differentiation capacity, and ability to repopulate a myeloablated
host. Assays such as the long-term culture-initiating cell
(LTC-IC)8 and extended LTC-IC assays9 measure
the ability of cells to generate myeloid progenitors after a prolonged
period in culture. However, these assays do not measure self-renewal, multilineage potential, or engraftment potential of progenitors. Although the recent development of the myeloid-lymphoid initiating cell
assay (ML-IC)10 and the self-renewing ML-IC assay (H. Liu and C.M.V., submitted) has allowed in vitro observation of multilineage differentiation and self-renewal of single cells, assessment of the
engrafting ability of human HSC requires transplant models. Several
xenogeneic models have been developed, including transplantation into
the humanized severe combined immunodeficiency (SCID)
mouse,11 into nonobese diabetic (NOD)-SCID
mice,12 the Beige-Nude-Xid (BNX) mouse13 and
preimmune fetal sheep.14,15 The engrafting human cell in
NOD-SCID or SCID mice has been defined as the SCID-repopulating cell
(SRC) or competitive repopulating unit (CRU), a cell that may be more
primitive than LTC-IC.11 In addition, several groups have
used autologous or allogeneic transplantation in rhesus or baboon
monkeys as a model for human stem cells.16,17 Each of these assays has its own advantages and disadvantages. How efficient they are at measuring human HSC and how they compare with in vitro assays that measure primitive progenitors is not known.
Ex vivo culture systems can be used to expand committed progenitors or
accessory cells.18,19 Here we focused on the use of ex
vivo expansion cultures to increase the number of long-term repopulating cells that have self-renewal and multilineage
differentiation capacity. Several ex vivo expansion systems have been
developed, including cytokine-rich stroma-free systems that may or may
not contain serum or systems in which hematopoiesis is supported by a
stromal feeder. The majority of studies in which progenitors are
cultured in stroma-free, serum-free, or serum-containing
cytokine-supplemented conditions show that culture for less than 6 days
results in a modest increase in SRC frequency, but loss of SRC if the
cultures are extended for longer periods.15,20,21 One
recent study from Piacibello and coworkers reported a more than 50-fold
expansion of SRC when UCB CD34+ cells were cultured for 6 to 10 weeks in a human serum-containing system with
fetal-liver-tyrosine-kinase-ligand (FL), thrombopoietin (Tpo), and/or
stem cell factor (SCF) and interleukin 6 (IL-6).22
A number of murine-derived stromal cell lines have been cloned that
support not only murine repopulating cells but also primitive human
hematopoietic progenitors.10,23-25 However, coculture of human cells with the murine feeder will not be suitable for clinical ex
vivo expansion. We developed noncontact cultures in which cells are
cultured in a transwell above a stromal feeder and have shown that
primitive LTC-IC, natural killer culture-initiating cell (NK-IC) and
ML-IC are maintained to the same or greater degree than when cells are
cocultured in contact with feeders.10,26 Because human
CD34+ cells remain separated from the murine feeder,
noncontact cultures avoid some of the practical problems associated
with contact cultures.
We describe here the ability of the murine fetal liver cell line,
AFT024, to maintain/expand human UCB CD34+ repopulating
cells assayed in the NOD-SCID mouse and the fetal sheep model. We found
that culture of UCB CD34+ cells for up to 28 days in an
AFT024 noncontact system supplemented with a combination of early
acting cytokines, including SCF, FL, Tpo, and IL-7, maintains
repopulating cells in NOD/SCID as well as fetal sheep. We provide the
first evidence that ex vivo expanded cells continue to contain
"long-term" repopulating cells because they can be serially
passaged to secondary NOD-SCID mice and secondary and tertiary fetal sheep.
Isolation of cells
Stromal feeder
Expansion cultures Noncontact cultures. Cells were plated in collagen coated transwells with a 0.4-µm microporous filter (Transwell-COL, Costar) above AFT024 feeders (noncontact) established in 6-well plates (Costar) as previously described.28 Initial cell concentration was 40 000 CD34+ cells/mL for 7-day cultures and 20 000 cells/mL for 14-day cultures. Individual wells contained 5 mL medium of which 2 mL contained the cells within the transwell. Medium consisted of RPMI supplemented with 20% FCS, 1000 U/mL penicillin, 100 U/mL streptomycin (Gibco-BRL), and 50 µmol/L 2-ME. Cytokine cocktails incorporated 10 ng/mL FL (Immunex, Seattle, WA), 10 ng/mL SCF (Amgen, Thousand Oaks, CA), 10 ng/mL IL-7 (R&D Systems, Minneapolis, MN), and/or 10 ng/mL Tpo (Amgen). Medium was added once at day 0 and changed every 7 days for 14- and 28-day cultures. MV8 stroma-free cultures.
Medium consisted of RPMI + 20% FCS with 250 pg/mL granulocyte
colony-stimulating factor (G-CSF) (Amgen), 200 pg/mL
macrophage-inflammatory protein-1 LTC-IC and colony-forming cell assays Thawed CD34+ cells (day 0) and CD34+ cell progeny from expansion cultures were plated in limiting dilutions in LTC-IC, as well as in colony-forming cell (CFC) assays. LTC-IC assays were performed as described.28,31 For LTC-IC assays, cells were plated in 4 dilutions of 22 replicates on preirradiated AFT024-coated 96-well plates. Medium consisted of Iscoves modified Dulbecco medium (IMDM) supplemented with 12.5% FCS, 12.5% horse serum (Stem Cell Technologies, Vancouver, BC, Canada), 1000 U/mL penicillin, 100 U/mL streptomycin, 2 µmol L-glutamine (Gibco-BRL), and 10 6 µmol hydrocortisone. Cultures were
maintained for 5 weeks with a half-medium change weekly. Medium was
then completely removed and replaced with clonogenic methylcellulose
medium consisting of 1.12% methylcellulose (Fisher, Chicago, IL),
IMDM, 30% FCS, 3 U/mL erythropoietin (Amgen), and supernatant of the
bladder cell carcinoma cell line 5637 (7.5%). After 2 weeks, wells
were evaluated for the presence or absence of hematopoietic colonies and scored as positive or negative, respectively. LTC-IC frequency was
then calculated according to Poisson statistics.
For CFC enumeration, 750 CD34+ cells or their progeny from expansion cultures were plated in methylcellulose containing IMDM supplemented with 30% FCS, 3 IU erythropoietin (Amgen), 10 ng/mL each of IL-3 (R&D Systems), G-CSF, and granulocyte-macrophage-CSF (GM-CSF; Immunex) as previously described.31 Cultures were incubated in a humidified atmosphere at 37°C and 5% CO2 and evaluated after 14 days for the presence of colony-forming units-granulocyte macrophage (CFU-GM) or burst forming units-erythroid (BFU-E). Transplantation experiments NOD-SCID recipients. A breeding colony of NOD-SCID mice was established from mice obtained from the Jackson Laboratories (Bar Harbor, ME). Mice were kept in specific pathogen-free conditions and maintained on acidified water and autoclaved food. Trimethoprim 60 mg and sulfamethoxazole 300 mg (Hoffmann-La Roche, Nutley, NJ) per 100 mL water was given twice weekly. At 6 to 8 weeks of age, mice were irradiated with 300 to 325 cGy at 57 cGy/min by a Mark 1 Cesium irradiator. Transplantation of UCB cells by tail vein injection occurred 24 hours after irradiation. Cell doses ranged from 25 to 150 × 103 CD34+ cells on day 0 (ie, uncultured) or the progeny of an identical number of cultured cells on days 7, 14, or 28. Six weeks after transplantation, mice were killed by cervical dislocation. Bone marrow (BM) was obtained by flushing femurs and tibias with IMDM 20% FCS. Cells from engrafted animals were then used for either secondary transplant experiments or extended phenotypes. When more than 2% human CD45+ cells were present in the murine marrow, cells from 2 femurs and 2 tibias were transplanted into individual secondary mouse recipients. Assessment of donor cell engraftment was by detection of the human- specific pan-leukocyte antigen CD45 (Becton Dickinson Immunocytometry Systems, San Jose, CA) conjugated to fluorescein isothiocyanate (FITC) or peridinin chlorophyll (PerCP). Three-color phenotyping was performed by staining cells with antihuman CD45 PerCP (Becton Dickinson), antimouse CD45 FITC (Pharmingen, San Diego, CA), and antihuman CD3 phycoerythrin (PE), CD14 PE, CD19 PE, CD33 PE, or CD34 PE (all from Becton Dickinson). Appropriate isotype controls were used. The frequency of the engrafting human cell in the mouse, defined as an SRC, was calculated by limiting dilution analysis. Mice were infused with increasing cell numbers and engraftment defined as detection of more than 0.5% human CD45+ cells. SRC frequency was calculated by Poisson statistics.12Fetal sheep recipients.
Freshly uncultured or cultured cells were suspended in IMDM + 20%
FCS, 10 ng/mL FL, SCF, and IL-7, and shipped overnight at room
temperature to the University of Nevada, Reno.32 Fifty (group X) to 100 (group I) × 103 uncultured
CD34+ cells or the progeny of an identical number of cells
cultured for 7 (group XI and II) or 14 (group XII and III) days were
injected into preimmune (day 57-62 of gestation) fetal sheep recipients using the amniotic bubble procedure as described (Figure
1).14,15 In some experiments, animals were killed 60 days
after transplantation and BM analyzed for human cells. The BM also
served as the source of CD45+ cells for transplant into
secondary recipients. Alternatively, animals were allowed to be born
and BM examined 6 months after transplantation. For secondary
transplants, human CD45+ cells were isolated from the BM of
primary recipients from groups I, II, and III at 60 days after
transplant by panning as described previously.14,15 The
CD45+ cells from the 2 animals from each group were pooled,
analyzed for CD34+ cell content, and injected into 3 secondary fetal recipients (group I, group IV, group II, group V, group
III, group VI). Animals were killed on day 60 after
transplantation and the BM cells were analyzed for the presence of
human cells. Marrow of these secondary recipients served as the source
of CD45+ cells transplanted into tertiary recipients.
Because the number of CD45+ cells was extremely low in the
secondary recipients in group VI, whole marrow was transplanted in
tertiary recipients (group IV, group VII, group V, group VIII, group
VI, group IX). All recipients were killed on day 60 after
transplantation and the BM cells were analyzed for the presence of
human cells.
For assessment of donor cell engraftment, BM MNC from the fetal and newborn sheep transplanted with human cells were analyzed for the presence of human cells by flow cytometry as previously described.32,33 Briefly, MNC were isolated from BM by hypotonic lysis of contaminating red cells. Antibodies specific for human CD3, CD20, CD33, CD34, CD45, and glycophorin A (Becton Dickinson) were conjugated to either FITC or PE. In each sample, 5 × 105 cells were labeled and expression of each antigen compared to the appropriate nonbinding isotype-matched control. Expression levels of 0.2% or more could be detected. In addition, BM MNC were cultured in methylcellulose (0.4-2 × 105 cells/mL) with supplemental erythropoietin (2 IU/mL), IL-3 (5 ng/mL), and GM-CSF (5 ng/mL) and human CFU-GM and CFU-Mix enumerated as described.32 Statistical analysis Results of experimental points obtained from multiple experiments are expressed as the mean ± SEM.
In a first series of experiments, we examined if culture in a
noncontact system using AFT024 as a stromal feeder supports UCB
CD34+ cells that engraft NOD-SCID mice. Freshly thawed UCB
CD34+ cells were transplanted in limiting dilutions into
NOD-SCID recipients. Alternatively, identical numbers of
CD34+ cells were cultured for 7 and 14 days in AFT024
noncontact cultures supplemented with FL, SCF, and IL-7, and progeny of
limiting dilutions of CD34+ cells transplanted in NOD-SCID
mice. The cytokine combination was chosen based on studies from our
group demonstrating that BM LTC-IC and NK-IC can be expanded under
these conditions10 (Table
1).
When transplanted with uncultured cells, 43 of 55 (78%) mice engrafted at levels of 0.5% to 26% (mean 6.5%). A higher mean engraftment level was noted with increasing cell doses although the frequency of engraftment did not change with increasing cell doses. The SRC frequency was 3.4/105 uncultured CD34+ cells (2.3-5.1, 95% confidence limits). When transplanted with cells cultured for 7 days, 41 of 55 (75%) mice engrafted at levels of 0.5% to 26% (mean 4.6%). The effect of cell dose on engraftment was not as apparent at this time point, although there was a trend toward a higher level of engraftment with increasing cell dose. The SRC frequency was 2.7/105 CD34+ cells (1.8-4.1, 95% confidence limits). When transplanted with cells cultured for 14 days, 34 of 43 (79%) mice engrafted at levels of 1.0% to 57% (mean 7.2%). No effect of cell dose on the level of engraftment was seen. The SRC frequency was 4.0/105 CD34+ cells (2.4-6.7, 95% confidence limits). Thus, SRC frequency as well as level of engraftment of UCB CD34+ cells is maintained following culture in an AFT024 noncontact system with FL + SCF + IL-7 for at least 14 days. We next compared the ability of AFT024 noncontact cultures supplemented
with FL, SCF, and IL-7 to support SRC with a similar system
supplemented with FL and Tpo. The latter combination is superior to an
FL, SCF, and IL-7 containing AFT024 noncontact system in expanding
primitive myeloid and lymphoid progenitors from UCB.26 We
also tested a stroma-free system as a preliminary step toward producing
a more clinically applicable system. Cultures were established and
after 7 and 14 days, we examined the number of nucleated cells,
CD34+ cells, CFC, LTC-IC, and SRC (Tables
2 and
3).
Culture of UCB CD34+ cells in AFT024 noncontact with FL, SCF, IL-7 produced a 76-fold increase in total nucleated cells (TNC) with associated 16-fold increase in CD34+ cells at day 14. Culture of UCB CD34+ cells in AFT024 noncontact with FL and Tpo yielded a 37-fold expansion in TNC and 8-fold expansion of CD34+ cells after 14 days. Culture in MV8 stroma-free cultures led to a 180-fold expansion of TNC by day 14, whereas CD34+ cells expanded only 2-fold. CFC and LTC-IC expansion was similar for all groups (Table 2). Mice were injected with 4 doses (25, 50, 75, and 100 × 103 cells) of uncultured UCB CD34+ cells. The mean engraftment levels were 13.1%, 9.0%, 4.7%, and 2.4%, respectively (Table 3). The SRC frequency was 3.8/105 uncultured CD34+ cells. The same doses of CD34+ were cultured for 7 and 14 days in each of the 3 culture conditions and progeny transplanted. Engraftment results after 7 and 14 days of culture in each of the conditions tested are summarized in Table 3. Consistent with what we describe above, the SRC frequencies in CD34+ cells cultured in FL, SCF, and IL-7 AFT024 culture conditions were 8.3/105 CD34+ cells and 3.6/105 CD34+ cells on days 7 and 14, respectively. SRC frequencies in FL and Tpo conditions were 3.2/105 CD34+ cells and 7.2/105 CD34+ cells on days 7 and 14, respectively. When cells were cultured in stroma-free cultures for 7 days, the SRC frequency was 1.2/105 CD34+ cells, and after 14 days, 6.1/105 CD34+ cells. Levels of human engraftment were similar for the 3 culture conditions. Studies from our laboratory indicate that expansion of primitive LTC-IC and NK-IC in AFT024-noncontact cultures supplemented with FL and Tpo is greater at 5 than 2 weeks.26 Similar results have been described by Piacibello and coworkers.34 We therefore tested whether engrafting cells could be maintained for longer periods in AFT024 noncontact culture with FL and Tpo or FL, SCF, and IL-7. After culture for 28 days, 3 of 5 mice transplanted with the progeny of 105 CD34+ UCB cells expanded in FL and Tpo engrafted at levels of 2%, 2%, and 3%. At a cell dose of 5 × 10,4 1 of 2 mice engrafted at a level of 3%. Two of 2 mice transplanted with the progeny of 105 cells expanded for 28 days in FL, SCF, and IL-7 engrafted at levels of 2% and 3%. To demonstrate that primitive progenitors engrafted, we examined if
multilineage engraftment was obtained and whether cells could be
serially passaged to secondary and tertiary recipients. No
CD3+ T cells were detected in animals that received either
day 0, day 7, or day 14 engrafted cells. However, 17.5% of human
CD45+ cells from day 0 engrafted cells were
CD14+, 48.8% CD19+, 42% CD33+,
and 13.8% CD34+ (Figure 2
and Table 3). Culture in an AFT024 noncontact system supplemented with
either FL, SCF, and IL-7, or FL and Tpo, or culture in a stroma-free
system resulted in a similar distribution of each marker.
We next tested whether human cells could be transferred to secondary and tertiary NOD-SCID mice. All BM cells obtained from 2 femurs and 2 tibiae of single primary NOD-SCID animals that had more than 2% human CD45+ cells were transplanted into single secondary NOD-SCID animals (1:5 dilution). Eighteen animals received cells from animals engrafted with uncultured CD34+ cells, 18 mice received cells from animals engrafted with 7-day AFT024 noncontact cultured cells, and 17 mice received cells from animals engrafted with 14-day AFT024 noncontact cultured cells, and 2 and 2 animals received cells from primary recipients transplanted with cells expanded for 7 and 14 days, respectively, in stroma-free MV8 conditions. Human CD45+ cells at levels between 0.6% and 2% were found in 4 of 8 animals that received cells from primary recipients engrafted with noncultured cells. Six of 18 (5 of 11 FL + SCF + IL-7 and 1 of 7 FL + Tpo) mice who received day-7 cells from AFT024 noncontact culture engrafted at levels of 1% to 5%. Eight of 17 mice (4 of 9 FL + SCF + IL-7 and 4 of 8 FL + Tpo) who received day 14 cells engrafted at levels between 0.5% to 20%. One percent human CD45+ cells were detected in the secondary recipient of cells recovered from one animal transplanted with day-7 MV8 stroma-free cultured CD34+ cells and no engraftment was seen in secondary recipients of day 14 MV8 stroma-free cultured CD34+ cells. Marrow from the 2 femurs and 2 tibiae from 9 secondary transplant recipients with more than 1% human CD45+ cells in the marrow were transplanted into tertiary NOD-SCID recipients. No engraftment was seen irrespective of the initial source of the cells. To further confirm these results and to evaluate if different in vivo
xenogeneic transplantation models measure similar cells, we also tested
the ability of uncultured and culture-expanded UCB CD34+
cells to engraft preimmune fetal sheep. Cells were cultured in AFT024
noncontact conditions with FL, SCF, and IL-7 for 7 and 14 days. In the
first set of experiments, pooled UCB was used to test the capacity of
the cultured cells to engraft in primary recipients and to be serially
passaged from primary to secondary and tertiary fetal sheep recipients.
The results of these experiments are summarized in Table
4. Fetal sheep transplanted with
105 uncultured CD34+ cells (group I) had
4.8% ± 0.7% human CD45+ cells in the marrow 2 months
after transplantation and this persisted for at least 6 months
(4.1% ± 0.5% human CD45+ cells). Fetal sheep
transplanted with day 7 cultured cells (group II) had 6.3% ± 1.4%
human CD45+ cells at 2 months and 4.3% ± 0.9% human
CD45+ cells at 6 months. Fetal sheep transplanted with
day-14 cultured cells (group III) engrafted at 5.6% ± 0.5% human
CD45+ cells at 2 months, but this fell to 0.2% ± 0.2%
human CD45+ cells at 6 months. A second experiment was done
in which 5 × 104 CD34+ cells, fresh or after
day 7 or day 14 of culture, from a single UCB were transplanted into
preimmune fetuses (groups X, XI, XII) (Table 4). Two months after
transplantation, animals were killed and human cell engraftment
evaluated. Again, similar levels of human cell engraftment were seen
for uncultured and ex vivo- expanded cells.
We also examined if human cells could be serially transplanted in
secondary and tertiary recipients as another measure for long-term
engrafting cells. Secondary and tertiary fetal sheep recipients were
evaluated 2 months after transplantation. Marrow of secondary (group
IV) and tertiary (group VII) recipients of uncultured cells had 5.2%
and 4.7% human CD45+ cells, respectively, at 2 months.
Marrow of secondary (group V) and tertiary (group VIII) recipients of
day-7 cultured cells had 4.4% and 0.8% human CD45+ cells,
respectively, at 2 months. Again, multilineage engraftment was seen
(Figure 3). Marrow of secondary
recipients of day-14 cultured cells (group VI) had less than 0.5%
human CD45+ cells and no human cells could be detected in
tertiary recipients of day-14 cultured cells (group IX). In all
engrafted animals, multilineage hematopoiesis was seen (Table
5).
Increased understanding of the regulatory processes involved in normal hematopoiesis has led to the development of a number of different culture systems aimed at expanding hematopoietic cell populations. A culture system that will expand HSC must increase the absolute number of primitive hematopoietic cells while maintaining their ability to produce multilineage progeny and repopulate myeloablated recipients. Several groups have developed stroma-free culture systems supplemented with multiple early acting cytokines. All but one study found limited expansion of cells capable of repopulating NOD-SCID mice or fetal sheep.15,20,21 Likewise, culture of progenitors on allogeneic human stromal feeders resulted in loss of engraftment capacity of both marrow and UCB CD34+ cells.35 Several stromal feeders have been cloned that support long-term repopulating cells from murine, human, and primate sources. For instance, irradiated baboons were rescued when transplanted with autologous marrow cultured on a porcine microvascular cell line for 7 days.17 When cocultured with the murine stromal cell line, AC6.21, human CD34+Thy-1+ cells continued to contain SRC.36 This suggests maintenance/expansion of at least short-term repopulating cells. Because studies were either in the autologous setting or without serial transplantation, it is not possible to assess whether HSC that can repopulate long-term were maintained/expanded. One drawback of these culture systems is that cells are cultured in contact with the stromal layer, which would not be clinically applicable. We have used a noncontact culture system as the basis for HSC
expansion, and used 2 in vivo transplantation models in which cells are
serially transferred to secondary and tertiary recipients to address
the question of expansion of long-term engrafting cells. We used the
AFT024 feeder, derived by Lemischka and colleagues from murine fetal
liver.27 AFT024 cells support maintenance of competitive
repopulating murine stem cells for more than 7 weeks as well as
growth of LTC-IC, E-LTC-IC, NK-IC, and
ML-IC.10,23,25,37 We have previously reported that
ex vivo culture of human UCB CD34+Lin To further demonstrate that the AFT024 noncontact system can
maintain/expand HSC, we transplanted uncultured and ex vivo- expanded
UCB CD34+ cells in preimmune fetal sheep. This also
provided us with the unique opportunity to compare the 2 xenogeneic
transplantation models. Although the NOD-SCID model may be the most
commonly used assay to measure engrafting human cells, a number of
studies suggest that serial transplantation to examine presence of
long-term repopulating cells is more easily achieved in the fetal sheep
than NOD-SCID model.15,39,40 Serial evaluation of marrow
of sheep recipients for more than 6 months to several years, which
cannot be done in the NOD-SCID model, is another way to measure
presence of long-term engrafting human cells. In addition, Civin and
colleagues have demonstrated that serial transfer of human cells to
secondary and tertiary fetal sheep recipients allows detection of the
most primitive progenitors. They showed that in contrast to
CD34+CD38 We also evaluated "long-term" engraftment. We examined sheep 6 months after transplantation or transfer of cells into secondary and tertiary fetal sheep recipients. Cells cultured ex vivo in AFT024 cultures supplemented with FL + SCF + IL-7 could support high levels of human hematopoietic cell engraftment 6 months after transplantation and could be serially transferred to secondary and even tertiary sheep recipients. Cells cultured for 7 days ex vivo produced multilineage engraftment in primary recipients at 6 months and also in secondary and tertiary fetal sheep recipients. These findings, taken together with the data from the NOD/SCID model, strongly suggest that multilineage engrafting cells that persist for at least 6 months are at a minimum maintained in AFT024 noncontact cultures. In view of the report by Civin and coworkers, the finding that engrafting cells could be transferred to secondary and tertiary recipients suggests that long-term engrafting cells can be maintained. The degree of engraftment of cells cultured ex vivo for 7 days in the tertiary recipients was lower than that of uncultured cells. This may indicate that the frequency of long-term engrafting cells has declined somewhat during culture. However, no decreased engraftment was seen in the primary recipients at 6 months or in the secondary recipients. Limiting dilutions will be needed to determine the importance of this observation. As we saw in the NOD-SCID model, day-14 expanded cells engrafted at 2 months in the fetal sheep model. Of note, serial transfer to secondary and tertiary sheep as well as assessment of the primary animals at 6 months showed that cells that could sustain human hematopoiesis for at least 6 months may decline significantly following 14 days of ex vivo expansion. This at first seems to contradict the results seen in the NOD-SCID model where no obvious differences were seen in secondary engraftment of day 7 and day 14 cultured cells. However, evaluation in the sheep model was at later time points than in the NOD-SCID model, as sheep are killed at 8 weeks rather than 6 weeks. Thus, our studies suggest that long-term engrafting cells may progressively decline with extended time in culture. To make this system more clinically suitable, we examined if we could develop a stroma-free system that would reproduce results obtained in AFT024 noncontact cultures. The stroma-free system was developed based on knowledge gained on growth factors and extracellular matrix components present in stroma-conditioned medium that are required for the support of LTC-IC.29,30,41,42 We have previously reported on the importance of specific 6-O-sulfated heparan-sulfate glycosaminoglycans for maintaining LTC-IC ex vivo. In addition, we have identified cytokines elaborated by human stromal feeders required for LTC-IC growth. We combined these components with 3 cytokines found to be present in nanogram concentrations in AFT024 supernatants (MCP-1, VEGF, and IL-8) (unpublished observation, 1999) and added a mixture of FL + SCF + IL-7 + Tpo. Consistent with previous studies from our group,29,41,42 a 2- to 3-fold LTC-IC expansion was seen that is similar to that seen in AFT024 noncontact cultures. Engraftment was seen with day-7 and -14 expanded cells and the frequency in day-14 cultured cells was 1.6-fold higher than in uncultured cells. Engrafted cells maintained multilineage differentiation capacity, and in 1 of 4 animals tested, we found secondary transfer of human cells. Whether these cells can engraft in the fetal sheep model and support hematopoiesis in secondary and tertiary sheep or for protracted periods of time in primary sheep is currently under investigation. However, these initial results suggest that it may be possible to develop defined stroma-free systems based on knowledge gained on factors involved in hematopoietic regulation by AFT024 that will support maintenance/expansion of long-term engrafting human cells. The variables involved in the engraftment of transplanted cells are not
well understood. In particular, the role of cell dose and accessory
cells in facilitating engraftment are variables we did not control in
this study. Thus, animals receiving cells at day 7 and day 14 received
much larger cell doses. Using a statistical model, van der Loo and
coworkers demonstrated a dose-response relationship of mobilized
peripheral blood CD34+ cells and
engraftment.43 Using BM
34+lin In conclusion, we show for the first time that cells capable of "long-term" engraftment in fetal sheep or NOD-SCID mice can be preserved when cultured for 7, and possibly 14 days, ex vivo in an AFT024 noncontact culture system or even a stroma-free system modeled on the AFT024 noncontact culture system. Culture methods described here should lead to the development of a clinically suitable culture system for ex vivo expansion, and allow for final demonstration that the conditions support long-term engraftment in human clinical trials.
Submitted May 30, 2000; accepted February 6, 2001.
Supported in part by: R01-DK-53673 (to C.M.V.), P01-CA-65493 (to C.M.V.), HL-52955 (to E.D.Z.), HL-49042 (to E.D.Z.), DK-51427 (to E.D.Z.), the Veterans Administration, and Fairview-University of Minnesota. C.M.V. is a Scholar of the Leukemia Society of America.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Catherine M. Verfaillie, Box 806 UMHC, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: verfa001{at}tc.umn.edu.
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© 2001 by The American Society of Hematology.
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Abstract
Introduction
(MIP-1
