Miharada et al. p.
1
Materials and methods
Cell culture
Human umbilical cord blood samples were purchased from the Cell Engineering
Division of RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). The use of human
umbilical cord blood was approved by the institutional review board of the RIKEN
Tsukuba Institute before this study was initiated.
CD34+ cells were isolated from human umbilical cord blood by supermagnetic
microbead selection using Direct CD34 progenitor isolation beads (Miltenyi Biotech,
Bergisch Gladbach, Germany) and MACS LS-columns (Miltenyi Biotech), and were
subsequently cultured. The culture procedure comprised four passages. In the first three
passages, cells were cultured in the presence of humoral factors as shown in Table 1 and
erythroid differentiation medium (EDM): StemSpan® H3000 (StemCell Technology,
Vancouver, BC, Canada) supplemented with 5% Plasmanate® Cutter (Bayer Japan,
Yodogawa, Osaka, Japan), -tocopherol (20 ng/ml; SIGMA, St Louis, MO, USA),
linoleic acid (4 ng/ml; SIGMA), cholesterol (200 ng/ml; SIGMA), sodium selenite (2
ng/ml; SIGMA), iron-saturated human transferrin (200 g/ml; SIGMA), human insulin
(10 g/ml; SIGMA), ethanolamine (10 M; SIGMA), and 2-mercaptoethanol (0.1 mM;
SIGMA).
In passage I, 1x105 CD34+ cells were cultured in 10 ml of EDM (1x104
cells/ml) either in the presence of human stem cell factor (SCF, 50 ng/ml; R&D systems,
Minneapolis, MN, USA), human erythropoietin (EPO, 6 IU/ml; KIRIN Brewery, Tokyo,
Japan), human interleukin-3 (IL-3, 10 ng/ml; R&D systems) (Protocol A) or in the
presence of SCF (50 ng/ml), EPO (6 IU/ml), IL-3 (10 ng/ml), human vascular
Miharada et al. p.
2
endothelial growth factor (VEGF, 10 ng/ml; R&D systems), and human insulin-like
growth factor-II (IGF-II, 250 ng/ml; R&D systems) (Protocol B) for six days. In
passage II, 3x105 cells, approximately 1/30 of cells expanded in passage I, were
cultured in 10 ml of EDM (3x104 cells/ml) in the presence of SCF (50 ng/ml) and EPO
(6 IU/ml) for four days. In passage III, 5x105 cells, approximately 1/50 of cells
expanded in passage II, were cultured in 10 ml of EDM (5x104 cells/ml) in the presence
of SCF (50 ng/ml) and EPO (2 IU/ml) for six days. In passage IV (enucleation step),
5x106 cells, approximately 1/10 of cells expanded in passage III, were cultured for four
days in 10 ml of enucleation medium (5x105 cells/ml): IMDM supplemented with 0.5%
Plasmanate® cutter, D-mannitol (14.57 mg/ml; SIGMA), adenine (0.14 mg/ml;
SIGMA), disodium hydrogenphosphate dodecahydrate (0.94 mg/ml; SIGMA), and
mifepristone (an antagonist of glucocorticoid receptor, 1 M; SIGMA). MAP described
in the text means the mixture of D-mannitol, adenine, and disodium hydrogenphosphate
dodecahydrate. The cultures were incubated at 37oC in 5% CO2 under humidified
conditions.
Viable cell number was assessed using an automated cell counter and an assay
based on the trypan blue dye exclusion method, ViCellTM (BECKMAN COULTER,
Fullerton, CA, USA).
Cell differentiation was monitored by microscopic morphology after
Wright-Giemsa staining. To evaluate the percentage of enucleated cells, all nucleated
and enucleated cells were separately counted in ten non-overlapping fields of the
microscope. The percentage of enucleated cells in the culture was calculated using the
formula: 100 x (number of enucleated cells/number of enucleated cells plus nucleated
Miharada et al. p.
3
cells).
Flow cytometry
Cells were stained as previously described1. The monoclonal antibodies (MoAbs) used
were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-human CD34 MoAb
(BD PharMingen, San Diego, CA, USA); allophycocyanin (APC)-conjugated
anti-human VEGF receptor-2 MoAb (BD Pharmingen); phycoerythrin (PE)-conjugated
anti-human CD221 (IGF-I receptor alpha) MoAb (R&D systems, Minneapolis, MN,
USA); FITC-conjugated anti-human CD71 (transferrin receptor) MoAb (eBioscience,
San Diego, CA, USA); PE-conjugated anti-human Glycophorin-A MoAb (BD
PharMingen); PE-conjugated anti-human Rh-D MoAb (Chemicon Australia, Melbourne,
Australia). Anti-human Fc receptor MoAb (Miltenyi Biotech) was used in all
experiments to inhibit nonspecific binding of MoAbs to Fc receptors. Cell viability was
monitored by propidium iodide (SIGMA) staining. Stained cells were analyzed by
FACS Calibur (BD Biosciences, San Jose, CA, USA). Flow cytometry data was
analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA) analysis software.
Reference
1. Nakamura, Y., Grumont, R.J. & Gerondakis, S. NF-
-Abl-induced
lymphoid transformation by functioning as a negative regulator of cyclin D1 expression.
Mol. Cell. Biol. 22: 5563-5574 (2002)
ACKNOWLEDGMENTS
We obtained human umbilical cord blood from the Cell Engineering Division of RIKEN
Miharada et al. p.
4
BioResource Center, which was supported by the National Bio-Resources Project and
the Stem Cell Resource Network (Banks at Miyagi, Tokyo, Kanagawa, Aichi, and
Hyogo) of the Ministry of Education, Culture, Sports, Science, and Technology in Japan
(MEXT), and from Dr. Ishiwata, I. (Ishiwata Hospital, Mito, Ibaraki, Japan). This work
was supported by grants from MEXT. We thank two anonymous reviewers for their
useful comments on previous drafts of this paper and all members in the Cell
Engineering Division for help, discussion, and secretarial assistance. Kenichi Miharada
was supported by the Junior Research Associate grant from RIKEN.