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Technical report
Role of different medium and growth factors on placental blood stem
cell expansion: an in vitro and in vivo study
M Berger
1, F Fagioli
1, W Piacibello
2, F Sanavio
2, K Mareschi
1, E Biasin
1, S Bruno
2,
L Gammaitoni
2, M Gunetti
2, F Nesi
1, E Madon
1and M Aglietta
21Department of Paediatrics, University of Turin Medical School, Turin, Italy; and2Institute for Cancer Research and Treatment, IRCC, Candiolo, University of Turin Medical School, Turin, Italy
Summary:
Expansion of haemopoietic stem cells from placental blood has been obtained with a combination of flt3 ligand (FL), thrombopoietin (TPO), kit-ligand (KL) with or without interleukin-6 (IL6) in serum-replete medium. For clinical use, cell expansion in the absence of serum is a clear advantage. Therefore, stem cell expansion in serum-free (SF) medium with a combi-nation of three (FL, TPO, KL) or four (FL, TPO, KL, IL6) growth factors was compared with the results obtained using fetal calf serum (FCS) or human serum (HS). Human CD34+placental blood cells were cultured
in the presence of FL, TPO, KLⴞ IL6 with SF medium,
HS and FCS for up to 8 weeks. CD34+, CFC, LTC-IC
content was measured at intervals. To determine the in
vivo repopulating capacity of expanded cells, CD34+
expanded cells were transplanted in sublethally
irradiated NOD/SCID mice. With the three growth fac-tor combination the CD34+cell number increased
stead-ily up to the 8 weeks of culture. CD34+ cells were
expanded 67.5-fold with SF, 11.7 with HS and 49.2 with FCS. However, when CFCs and LTC-ICs were con-sidered, a continuous expansion was observed only with HS and FCS, whereas in SF medium after 6 weeks their number started to decline. The addition of IL-6 did not change the expansion significantly. Cells grown ex vivo for 14 days were transplanted into NOD/SCID mice. The engraftment of human cells in mice was higher for serum-replete than for SF expanded cells. Nevertheless, SF cultured cells were also able to engraft both marrow and spleen in all animals. In addition, engrafted human cells still maintained clonogenic ability. With KL, FL, TPOⴞ IL6 it is possible to expand haemopoietic pro-genitor cells in a SF medium. Compared with serum-replete cultures, the absolute number of clonogenic cells and in vivo repopulating cells is lower. Although the degree of expansion remains significant, a clinical trial still needs to be carried out to address the question of Correspondence: Dr F Fagioli, Dipartimento di Scienze Pediatriche e dell’Adolescenza, Ospedale Infantile Regina Margherita, Piazza Polonia 94, 10126 Torino, Italy
Received 6 September 2001; accepted 2 January 2002
whether this expansion might be useful in reducing post-transplant aplasia.
Bone Marrow Transplantation (2002) 29, 443–448. DOI:
10.1038/sj/bmt/1703390
Keywords: placental blood; long-term culture;
serum-free expansion; NOD/SCID mice
Human placental blood is a source of haemopoietic stem cells both for transplantation1–4 and gene therapy
appli-cations.5 However, considerable interest in developing
methods for expanding cord blood stem cell numbers in
vitro6–14has been stimulated by the fact that a single cord
blood collection may not be sufficient to guarantee engraftment of adult allogeneic recipients. The cultures are traditionally performed in a medium containing fetal calf serum (FCS) but to utilise expanded cells in clinical prac-tice, ‘good manufacturing practice’ (GMP) standards and serum-free (SF) conditions are required. In particular, FCS must be excluded from the medium because it may contain allergenic substances and transmissible infections. Further-more, pooled human serum (HS) should be excluded to eliminate variables linked to individual donors.15,16
In this study we investigated how CD34+placental blood cells grow in SF conditions in the presence of haemopoietic growth factors (flt3 ligand (FL), thrombopoietin (TPO), kit ligand (KL) ⫾ interleukin 6 (IL6)) but in the absence of stroma. At different times, total cell, CD34+, CFC and LTC-IC content was measured. Moreover, after 14 days expansion the in vivo repopulating capacity of cells was determined. The results were compared with those obtained in the presence of HS or FCS.
Materials and methods
CD34+cell purification
Twenty-four placental blood samples were obtained, with the informed consent of the mother, from full-term deliver-ies. CD34+ cells were isolated from the placental blood mononuclear fraction using a MiniMACS CD34 isolation kit (Miltenyi Biotech, Bisley, UK) according to the
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facturer’s instructions. The efficiency of purification was verified by flow cytometry counterstaining with an anti-CD34-phycoerythrin (PE) antibody (HPCA-2; Becton Dickinson, San Jose, CA, USA).
Liquid cultures
A total of 2 ⫻ 104 initial CD34+ cells were cultured in
quadruplicate in flat bottomed 24-well plates in 1 ml of Iscove’s modified Dulbecco’s medium (IMDM; GIBCO BRL, Grand Island, NY, USA) supplemented with 10% FCS (Hyclone, Logan, UT, USA), 10% HS or SF con-ditions containing bovine serum albumin (BSA; Sigma Chemical Co, Milan, Italy) 20 mg/ml, insulin 10g/ml and transferrin 200l/ml (BIT; Stem Cell Technologies, British Columbia, Canada). The following growth factors were added: rhFL (50 ng/ml) + rhTPO (20 ng/ml) + rhKL (50 ng/ml) (three GF combination); rhFL (50 ng/ml)+ rhTPO (20 ng/ml)+ rhKL (50 ng/ml) + rhIL6 (10 ng/ml) (four GF combination). Each growth factor was added twice a week to the cultures. The wells were maintained at 37°C during culture. At the initiation of culture the number of CFC con-tent in 1 ml was determined by triplicate plasma clot assays. Each week half of the medium was removed and replaced with fresh medium and growth factors. The removed cells were counted and assayed for CFC, LTC-IC content and analysed for CD34+phenotype. For NOD/SCID mice repo-pulating experiments, CD34+were grown both in 25 or 75 cm2flasks with IMDM or X-vivo 15 (Bio-Whittaker,
Walk-ersville, MD, USA) medium for 14 days. Weekly cultures were not demipopulated but medium was added in order to double the volume. Aliquots were removed from the medium and the cells were counted and analysed for CD34+, CFC and LTC-IC content.
Recombinant human cytokines
Recombinant human (rh) kit-ligand (rhKL) and recombi-nant human thrombopoietin (rhTPO) were generous gifts from Amgen (Thousand Oaks, CA, USA), recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human interleukin-3 (rhIL-3) and recombinant human interleukin-6 (rhIL-6) were from Sandoz (Basel, Switzerland), recombinant human Flt3 ligand was kindly provided by SD Lyman (Immunex Corp, Seattle, WA, USA), recombinant human erythropoietin (rhEPO, Eprex) was from Cilag (Milan, Italy).
Flow cytometric analysis
Initial and cultured cells were evaluated for the expression of CD34 antigen. Cells were labelled with monoclonal anti-bodies anti-CD34 PE (HPCA-2; Becton Dickinson, San Jose, CA, USA). Cells were enumerated according to ISH-AGE guidelines.17Labelling lasted for 30 min in the dark,
at 4°C. Then, cells were washed twice in phosphate-buff-ered saline (PBS)+ 1% BSA and analysed with a FACScan flow cytometer (Becton Dickinson). At least 5000 events were acquired for each analysis. When fluorescent cells rep-resented only a minority of the total population, many more
events were considered (20 000). Analysis was performed with Cell Quest software (Becton Dickinson).
Haemopoietic cell cultures
Assays for CFC (GM, BFU-E, GEMM, CFU-Meg) were performed as follows. For CFU-GM 1 ⫻ 103
initial CD34+ cells and suitable aliquots of liquid culture were cultured in four dishes per point in 3% agar, 15% FCS in IMDM. For CFU-Meg the same number of cells was cultured in plasma clot assays (four dishes per point). For BFU-E and CFU-GEMM the same number of cells was cultured in 1.3% methylcellulose (Fluka, Chemika Bio-chemika, Buchs, Switzerland) and IMDM containing 30% FCS at 37°C in a humidified atmosphere at 5% CO2in air.
Colony scoring was performed on day 12 for CFU-Meg (at the immunofluorescent microscope after staining with a FITC (fluorescein–isothiocyanate)-conjugated anti-human GPIIb/IIIa antibody) and on day 14 for CFU-GM, BFU-E and CFU-GEMM. To sustain colony formation several growth factors were added: for BFU-E and CFU-GEMM, rhIL-3 (20 ng/ml), rhGM-CSF (10 ng/ml), rhEPO (3 U/ml) and rhKL (50 ng/ml) were added. For CFU-GM, rhIL-3 (20 ng/ml), rhGM-CSF (20 ng/ml) and rhKL (50 ng/ml) were added. For CFU-Meg, rhIL-3 (5 ng/ml) was the only growth factor added.
Long-term culture initiating cell cultures
The LTC-IC content of the cell suspension was determined by limiting dilution as previously described.9–18 In short,
10 to 1000 initial CD34+ cells or suitable aliquots during liquid cultures were washed and plated on pre-established bone marrow stroma as previously described. Cultures were maintained for 5 to 6 weeks at 37°C after which cells were harvested and seeded in a methylcellulose medium with 30% FCS, rhEPO (3 U/ml), rhKL (50 ng/ml), rhIL-3 (20 ng/ml), rhGM-CSF (20 ng/ml) and rhG-CSF (20 ng/ml). These cultures were incubated at 37°C at 5% CO2 in air.
The LTC-IC number was obtained by CFC output number in limiting dilution assay.
Bone marrow stroma
Human bone marrow was obtained by aspiration from the posterior iliac crests of healthy bone marrow donors, after informed consent. The mononuclear fraction was isolated using Ficoll–Hypaque (Nyegaard, Oslo, Norway) density centrifugation. In short, cells were seeded at 10⫻ 106/cm2
in 25 cm2flasks in the presence of 12.5% FCS, 12.5% horse
serum (Hyclone), 5 ⫻ 10−5m 2-mercaptoethanol (Sigma, St Louis, MO, USA), 1⫻ 10−6m hydrocortisone (Sigma) and penicillin/streptomycin. The medium was replaced twice weekly until confluence. Later, LTC-IC cultures were grown on a mixed murine fibroblastoid cell line (M210B4 and SL/SL) engineered to produce KL+ IL3 + G-CSF.
Animals
Twenty NOD/Ltsz scid/scid (NOD/SCID) mice were obtained from Charles River Italia (Calco, Italy) and
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tained in the animal facilities at CIOS (Turin, Italy). All the animals were handled under sterile conditions and maintained in microisolator cages. The mice to be injected were exposed at 6–8 weeks of age to 350 cGy of total body irradiation with a137Cs source. Within 24 h the mice were
injected with placental expanded cells with a single intra-venous injection. The mice were killed 5–6 weeks later. Controls were transplanted with 30 000 CD34+ non-expanded cells.
Analysis of human cells in murine tissue
Bone marrow cells were flushed from femurs and tibias of each mouse using a syringe and a 26-gauge needle. Cells were resuspended at 2 ⫻ 106cells/ml in Hank’s balanced
salt solution (HBSS; GIBCP BRL) containing 0.1% BSA and 0.01% sodium azide and incubated with mouse IgG (Fluka, Chemika Biochemika) and 5% human serum to block non-specific binding. The cells were then washed with PBS and incubated for 20 min with an anti-human CD45 monoclonal antibody, and simultaneously stained with an anti-human CD34, CD19, CD3, Glyco A and CD33 monoclonal antibodies (Becton Dickinson). Some cells in each condition were incubated with irrelevant monoclonal antibodies conjugated FITC or PE to exclude non-specific binding. Cells from unmanipulated mice were also stained with each monoclonal antibody used to test human engraftment. Only levels of fluorescence that excluded 99.9% of these negative controls were considered as specific.
Statistical analysis
Results are expressed as medians. A single hit was applied for LDA Poisson statistics. The frequency of LTC-IC in the cell suspension was calculated using a maximum likelihood estimator. Statistical differences among groups were calcu-lated by Student’s t-test as per P⬍ 0.05.
Results
Ex vivo expansion of human placental CD34+cells
In an effort to induce long-term proliferation and amplifi-cation of primitive haemopoietic progenitors contained in CD34+ placental cells, stroma-free suspension cultures stimulated by the early acting cytokines FL, TPO, KL, and ⫾ IL6 were set-up. These growth factors were used with three different media (SF medium and serum-replete medium (HS and FCS)) in order to identify the influence of serum on cell growth.
Total cell number and CD34+output: The cell and CD34+
populations underwent continuous expansion in SF, HS and FCS media with comparable values. For SF conditions we had a 1780-fold increase for total cell expansion compared to 3840 for HS and 1472 for FCS, respectively, after 8 weeks culture. For CD34+cell expansion, SF gave rise to a 67.5-fold CD34+increase compared to 11.7 for HS and
10 000 1 000 100 10 1 0.1 100 10 1 0.1 1000 100 10 1 0.1 100 10 1 0.1 Start 1 2 4 6 8 Start 1 2 4 6 8 Start 1 2 4 6 8 Start 4 6 8 Weeks of culture Fold increase Cells CD34+ CFCs LTC-ICs Serum free Human serum Fetal calf serum
Serum free Human serum Fetal calf serum
Serum free Human serum Fetal calf serum
Serum free Human serum Fetal calf serum
a
b
c
d
Figure 1 Comparative expansion of 2⫻ 104CD34+placental cells.
Out-put of cells, CD34+, CFCs and LTC-ICs with FL (50 ng/ml), TPO (20
ng/ml), KL (50 ng/ml) in liquid cultures containing serum-free medium, 10% human serum, or 10% fetal calf serum. Cells were grown in Iscove’s modified Dulbecco’s medium. The results represent a median fold cumu-lative increase of six experiments.
49.2 for FCS after 8 weeks of culture (Figure 1a, b). Table 1 shows phenotypic analysis of the three growth factor combination in a serum-free condition of placental blood CD34+expansion after 14 days’ culture.
CFC and LTC-ICs output: CFC expansion was similar in
all culture conditions (Figure 1c). The marginally lower output of CFC in SF conditions was not significant. LTC-IC behave rather differently since their number continued to increase in the presence of serum up to the 8th week of culture, whereas in a SF condition their number declined after the 6th week of culture (Figure 1d). IL6 did not
sig-Table 1 Phenotypic analysis of CD34+ expanded cells with three growth factor combination after 14 days culture
3 GFs % CD34+ 11.1 CD34+/CD38− 3.7 CD13+ 43 CD33+ 13 CD14+ 11.7 CD41+ 5.1
446
nificantly affect the growth pattern with or without serum (Figure 2a, b, c, d).19
Analysis of NOD/SCID mice repopulating cell expansion in different medium
To investigate NOD/SCID mice repopulating cell expan-sion, groups of sublethally irradiated NOD/SCID mice were injected with CD34+ expanded cells. In these series of experiments the four growth factor combination was used and two different media (IMDM and X-vivo 15) were com-pared. Cells were expanded for 14 days and then injected into NOD/SCID mice. Controls were transplanted with 30 000 non-expanded cells. Six to 8 weeks later, bone mar-row and spleen cells of animals that had been killed were assessed for human haemopoietic engraftment. As reported in Table 2, higher levels of human engraftment in cultures containing HS (33%) or FCS (65%) were found. Neverthe-less, SF cultured cells were also able to engraft both mar-row and spleen in all animals. In particular, engraftment was higher with IMDM (up to 30% of human engraftment as a median of three experiments, range from 20 to 47%) than with X-vivo 15 medium (8.4%, range from 5.3 to
10 000 1 000 100 10 1 0.1 100 10 1 0.1 1000 100 10 1 0.1 100 10 1 0.1 Start 1 2 4 6 8 Start 1 2 4 6 8 Start 1 2 4 6 8 Start 4 6 8 Weeks of culture Fold increase Cells CD34+ CFCs LTC-ICs Serum free Human serum Fetal calf serum
Serum free Human serum Fetal calf serum
Serum free Human serum Fetal calf serum
Serum free Human serum Fetal calf serum a
b
c
d
Figure 2 Comparative expansion of 2⫻ 104CD34+placental cells.
Out-put of cells, CD34+, CFCs and LTC-ICs with FL (50 ng/ml), TPO (20
ng/ml), KL (50 ng/ml) and IL-6 (10 ng/ml) in liquid cultures containing serum-free medium, 10% human serum or 10% fetal calf serum. Cells were grown in Iscove’s modified Dulbecco’s medium. The results rep-resent a median fold cumulative increase of six experiments.
11%). Interestingly, higher marrow engraftment correlates with spleen engraftment in all conditions and all haemopo-ietic lineages were found among murine tissues. Character-istically, there was higher myeloid cell engraftment in mar-row and lymphoid cells in spleen in all conditions analysed. Human CD34+cells were found in both marrow and spleen cells. Higher CD34+cell engraftment was found with FCS-replete medium (4.8%). Once again, the SF condition also ensured CD34+ cell engraftment (3.6% and 1.8% with IMDM or X-vivo 15, respectively). Unexpectedly, human cell engraftment did correlate with CFC but not with the LTC-IC content. In control mice human engraftment was very low (0–1.2%).
Discussion
The ability to expand haemopoietic cells under conditions that allow the retention of their functional characteristics when reinfused holds great promise. However, optimal defined conditions for human haemopoietic stem cell expansion need to be carefully developed. Our group recently reported in vitro and in vivo expansion of placental CD34+ cells cultured with FL+TPO ⫾ KL ⫾ IL6 in a serum-replete medium, demonstrating that expansion for 4 to 10 weeks involved NOD/SCID mice repopulating cells.9,10Since SF conditions are essential for clinical
devel-opment, the present experiments were planned.
In the present study, CD34+placental blood cells gave rise to an up to 90-fold increase of CFCs when cultured for 8 weeks in SF medium added to FL, TPO and KL. Furthermore, CD34+ cells were expanded up to 50-fold over the initial seeding level. These data are not signifi-cantly different from those obtained in the presence of FCS, and are consistent with previous studies using long-term ex
vivo culture of human cells in serum-replete media.7
There-fore, the data support the feasibility of using SF medium in such studies. The kinetics of LTC-IC amplification were comparable among samples cultured with SF medium and serum-replete medium until the 6th week of culture, after which, however, a decline was observed. IL-6 did not sig-nificantly improve the cell, CFC and CD34+expansion in our culture system, neither did it change LTC-IC outcome. Given the mixed in vitro results where LTC-IC declined in SF conditions, it became relevant to assess whether the in
vivo repopulating capacity of NOD/SCID mice was
main-tained. Our data indicate that CD34+cells cultured in the presence of FL, TPO, KL and IL-6 for up to 14 days engraft 100% of the irradiated mice. These data suggest that SF culture conditions can support both short-term and long-term marrow repopulating cells. These results demonstrate unequivocally that human haemopoietic stem cell activity assessed by their ability to regenerate lymphoid and myeloid progeny after 6 to 8 weeks in irradiated NOD/SCID mice is not inevitably or irreversibly lost when cells are activated mitogenically with soluble growth fac-tors.20 In addition, our findings show that most, if not all,
of the stem cells present in the CD34+fraction of human placental blood can be stimulated to proliferate rapidly in
vitro by this growth factor combination.21Another
LTC-447 Table 2 Engraftment of placental blood stem cells expanded with FL, TPO, KL, IL6 in media containing serum or serum substitute for 14 days
Expansion for 14 days with KL, FL, TPO and IL6
Ex-vivo 15+ FCS IMDM+ FCS Ex-vivo 15+ HS IMDM+ HS Ex-vivo 15+ SF IMDM+ SF
Cell fold increase 217 225 175 203 101 120
CD34+fold increase 5.5 2.5 3.8 2.1 0.4 0.69
CFC fold increase 13 43.5 11.5 34.2 11.7 22.3
LTC-IC fold increase 9.6 6.78 4.96 5.4 4.4 12.7
Marrow Spleen Marrow Spleen Marrow Spleen Marrow Spleen Marrow Spleen Marrow Spleen
% % % % % % % % % % % % CD45+ 17.5 1.05 64.9 6.3 15.9 1.2 33.4 7.1 8.4 1.24 31.9 4.26 CD34+ 2.4 0.25 4.89 1.93 1.85 0.11 2.55 2.05 1.8 0.35 3.6 1.3 CD19+ 4.6 0.42 16.9 3.45 4.12 0.65 13.7 4.94 3.9 0.95 10.3 3.8 CD33+ 12.8 0.36 43.9 2.21 10.45 0.21 18.5 2.56 4.3 0.05 20.9 1.1 CD3+ 0.2 0.01 0.29 0.17 0.25 0 0.42 0.2 0.42 0.2 0.28 0.08 Glyco-A+ 0.15 0.06 1.1 0.18 0.07 0.1 0.14 0.04 0.05 0.01 0.84 0.16
The percentage of human cell engraftment refers to all bone marrow and spleen cells. The results represent the median fold increase of three experiments.
IC and in vivo repopulating activities. However, other examples of a dissociation in LTC-IC and function in murine cells have been reported, indicative of differences in the molecular mechanisms required for primitive cells to be detected in these two assays.15,20,21
Taken together, these findings confirm the concept of a significant persisting population of expanded transplantable stem cells in cultures of human placental blood cells stimu-lated by FL, TPO, KL⫾ IL6.
The demonstration that SF cultures support a significant expansion of progenitor cells might represent an important alternative to the use of serum and provides a basis for further studies on the transplantation of human haemo-poietic expanded stem/progenitor cells.
Acknowledgements
We are grateful to Andrew M Garvey BAHons LTCL (TESOL) for editorial assistance. This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano), from the Ministero dell’Universita` e della Ricerca Scien-tifica e Tecnologica (MURST, Roma) to WP, EM, MA and from the Associazione Donatrici Italiane di Sangue di Cordone Ombel-icale (ADISCO, Sezione Piemonte).
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