First published online March 30, 2004
doi: 10.1242/10.1242/dev.01065
Development 131, 1869-1879 (2004)
Published by The Company of Biologists 2004
Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro
Katsutsugu Umeda1,
Toshio Heike1,
Momoko Yoshimoto1,
Mitsutaka Shiota1,
Hirofumi Suemori2,
Hong Yuan Luo3,
David H. K. Chui3,
Ryuzo Torii4,
Masabumi Shibuya5,
Norio Nakatsuji2 and
Tatsutoshi Nakahata1,*
1 Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54
Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
2 Department of Development and Differentiation, Institute for Frontier Medical
Science, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507,
Japan
3 Department of Medicine, Boston University School of Medicine, 88 East Newton
Street, Boston, MA 02118, USA
4 Research Center for Animal Life Science, Shiga University of Medical Science,
Tsukinowa-cho, Seta, Ohtsu, Shiga 520-2192, Japan
5 Division of Genetics, Institute of Medical Science, University of Tokyo, 4-6-1
Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
View larger version (35K):
[in a new window]
|
Fig. 1. Development of primitive and definitive erythrocytes. Primate ES cells
(4x103 cells/well) were cultured onto OP9 stromal cells for 6
days. The induced cells were harvested and re-cultured at a concentration of
1x105 cells per well onto fresh OP9 stromal cells, with or
without 10 U/ml EPO. The floating cells were harvested every other day and
analyzed by May-Giemsa staining and immunostaining against human hemoglobin
(Hb), fetal hemoglobin (HbF) and embryonic hemoglobin (HbEmb). (A-E) Day 12;
(G-J) day 18. (A,F) May-Giemsa staining of erythrocytes. (B,G) Hb (FITC) and
HbF (Cy3) staining of erythrocytes. (C-E,H-J) Hb (FITC) and HbEmb (Cy3)
staining of erythrocytes. Merged images are shown in B,E,G and J. Nuclei were
labeled with Hoechst 33342 in B-E and G-J. Scale bars: 10 µm. (K)
Sequential analysis of the number of erythrocytes, with (black circles) or
without (white circles) EPO. (L) Sequential analysis of the proportion of
definitive erythrocytes (EryD) among total erythrocytes, with (black columns)
or without (white columns) EPO. EryD were defined as Hb- and HbF-positive,
HbEmb-negative erythrocytes, whereas primitive erythrocytes were Hb-, HbF- and
HbEmb-positive. Data represent the mean±s.d. of triplicate wells.
Representative results from one of three independent experiments are
shown.
|
|
View larger version (52K):
[in a new window]
|
Fig. 2. Development of megakaryocytes and myeloid cells. (A,B) May-Giemsa staining
(A, left cell) and CD41 (Cy3) staining (B) of megakaryocytes on day 12. Nuclei
were labeled with Hoechst 33342 in B. (C,D) May-Giemsa staining (C) and
myeloperoxidase staining (D) of myeloid cells on day 18. Scale bars: 10 µm.
(E) Sequential analysis of the percentages of erythrocytes (white columns),
myeloid cells (striped columns) and megakaryocytes (black columns) among total
hematopoietic cells in the presence of EPO. Each column represents the mean of
triplicate wells. Representative results from one of three independent
experiments are shown.
|
|
View larger version (53K):
[in a new window]
|
Fig. 4. Development of adherent hematopoietic cell clusters. (A,B) An adherent
hematopoietic cell cluster (A) and a cluster with a cobblestone appearance
that grew underneath the OP9 stromal cells (B). (C) Alkaline phosphatase
staining of adherent hematopoietic cell clusters immunolabeled with human CD34
antibody. Scale bars: 100 µm.
|
|
View larger version (53K):
[in a new window]
|
Fig. 5. Development of hematopoietic colonies. Methylcellulose colony-forming
assays were performed on day 8. Using adherent cells dissociated with trypsin
treatment, only a few GM colonies were observed (A,B). When the medium was
replaced with methylcellulose-containing medium, erythroid colonies were
generated on the OP9 stromal cell layer (C-F). (A,C) Morphology of a GM colony
(A) and a primitive erythroid colony (C). (B,D) May-Giemsa staining of a GM
colony (B) and a primitive erythroid colony (D). (E,F) Human hemoglobin (Hb;
FITC) and fetal hemoglobin (Cy3; E), and Hb (FITC) and embryonic hemoglobin
(Cy3; F) staining of erythrocytes in a primitive erythroid colony. Nuclei were
labeled with Hoechst 33342. Merged images are shown. Scale bars: 100 µm in
A,C; 10 µm in B,D-F. (G) RT-PCR analysis of globin gene expression in
erythroid colonies. M, size marker; BM, adult cynomolgus monkey bone marrow
cells; K, human erythroblastic cell line K562; O, OP9 stromal cells; EC1-EC3,
primitive erythroid colonies. Seven other individual erythroid colonies showed
similar globin gene expression patterns.
|
|
View larger version (38K):
[in a new window]
|
Fig. 6. Sequential flow cytometric analysis of KIT, FLK1 and CD34 during
differentiation induction. Undifferentiated ES cells, or the cultures on day
4, 6, 12 and 18, were stained with antibodies specific for KIT, FLK1 or CD34,
or with control IgG1. Plots show the isotype control IgG1 staining profile
(dashed lines) versus the specific antibody staining profiles (solid lines).
Representative results from one of three independent experiments are
shown.
|
|
View larger version (30K):
[in a new window]
|
Fig. 7. Sequential RT-PCR analysis of genes associated with hematopoietic
development in cultures during liquid culture differentiation. M, size marker;
BM, adult cynomolgus monkey bone marrow cells; K, human erythroblastic cell
line K562; ES, undifferentiated ES cells; ME, mouse embryonic fibroblasts; O,
OP9 stromal cells; d, days after the induction of differentiation in the
presence of EPO.
|
|
View larger version (41K):
[in a new window]
|
Fig. 8. Effects of EPO and various growth factors on primitive and definitive
hematopoiesis. Primate ES cells were cultured for 6 days with VEGF, bFGF or
BMP4. The induced cells were harvested and re-cultured with (black circles) or
without (white circles) 10 U/ml EPO. The floating cells were analyzed as
described in Fig. 2. Adherent
clusters ( 20 cells) were counted on day 10. Primitive erythroid colonies
(50 cells) were counted 7 days after replacing the medium with
methylcellulose-containing medium on day 8. (A,C) Effects of EPO and various
concentrations of VEGF (0 ng/ml, circles; 5 ng/ml, triangles; 10 ng/ml,
squares; 20 ng/ml, diamonds) on the number of erythrocytes (A) and total
hematopoietic cells (C). (B) Sequential analysis of the proportion of
definitive erythrocytes (EryD) among total erythrocytes in the presence of EPO
alone (black columns), or EPO plus VEGF (20 ng/ml; striped columns). (D)
Sequential analysis of the percentages of erythrocytes (white columns),
myeloid cells (striped columns) and megakaryocytes (black columns) among total
hematopoietic cells in the presence of EPO plus VEGF (20 ng/ml). (E,F) Effects
of EPO and growth factors on the number of adherent hematopoietic clusters (E)
and primitive erythroid colonies (F). E, EPO; V, VEGF (5 to 20 ng/ml); b, bFGF
(20 ng/ml); B, BMP4 (20 ng/ml). (G,H) Effects of bFGF (20 ng/ml, triangles),
BMP4 (20 ng/ml, squares) and VEGF (20 ng/ml, diamonds) on the number of
erythrocytes (G) and total hematopoietic cells (H). Data represent the
mean±s.d. of triplicate wells. Representative results from one of three
independent experiments are shown.
|
|
View larger version (47K):
[in a new window]
|
Fig. 9. More efficient primitive and definitive hematopoiesis is induced by
re-plating sorted CD34-positive cells. (A-C) Flow cytometric analysis and cell
sorting of cultures on day 10 with human CD34 antibody. A sorting gate in B
was defined according to the intensity of staining with an isotype control
antibody (A). Reanalysis of the sorted cells confirmed the purity as 95 to 98%
(C). (D) Large adherent hematopoietic cell cluster with a cobblestone
appearance on day 25. Scale bars: 100 µm. (E-G) May-Giemsa staining (E),
human hemoglobin (Hb; FITC) and embryonic hemoglobin (Cy3; F), and Hb (FITC)
and fetal hemoglobin (Cy3; G) staining of day 25 erythrocytes grown in the
presence of EPO. Nuclei were labeled with Hoechst 33342. Merged images are
shown. Scale bars: 10 µm. (H,I) Sequential analysis of the number of
erythrocytes (H) and total hematopoietic cells (I), with (black circles) or
without (white circles) 10 U/ml EPO. (J) Sequential analysis of the proportion
of definitive erythrocytes among total erythrocytes. (K) Sequential analysis
of the percentages of enucleated erythrocytes (stippled columns), nucleated
erythrocytes (white columns) and myeloid cells (striped columns) in the
presence of EPO. Data represent the mean±s.d. of triplicate wells.
Representative results from one of three independent experiments are
shown.
|
|
© The Company of Biologists Ltd 2004
and 
), fetal
(
) and adult (ß and 
) globin expression in erythrocytes
during liquid culture differentiation. M, size marker; BM, adult cynomolgus
monkey bone marrow cells; K, human erythroblastic cell line K562; ES,
undifferentiated ES cells; ME, mouse embryonic fibroblasts; O, OP9 stromal
cells; d, days after the induction of differentiation in the presence of
EPO.