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Hematopoiesis is the term
applied to the myriad processes resulting in blood cell production. The
description of hematopoietic stem cell activity stands as a tremendous
scientific milestone of the last century and was a culmination of many
studies. Moreover, the detailed analysis of hematopoiesis also directly
facilitated the development of a profoundly beneficial medical intervention,
namely bone marrow transplantation, a stunning example of the key role
that basic research plays in improving human health.
Our laboratory has a keen
interest in understanding the process of hematopoiesis at all levels including
the specification of blood tissue in the developing embryo and mechanisms
contributing to efficient transplantation of blood-forming activity. Our
work in hematopoiesis bridges our interests in
PLURIPOTENT
CELL BIOLOGY
and
CANCER
THERAPEUITICS
with projects in the derivation
and engraftment of lympho/myeloid repopulating activity from an ES cell
starting point as well as the study of disease-specific stem cells in
the context of blood formation.
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A
composite depicting key events in hematopoiesis in vivo for the mouse (on
top and scaled in days) and human (on bottom and scaled in weeks). Both
murine and human figures are interpreted using the same key as follows:
vertical red dashed line at left, onset of circulation; vertical green solid
line at right, birth; EryP, primitive nucleated erythroblasts in peripheral
blood (PB); EryD, definitive anucleate erthrythrocytes in PB; P-Sp, onset
of hematopoiesis in the paraaortic splanchnopleura (down-slanting hash marks);
AGM, onset of hematopoiesis in aorta-gonad-mesonephros (up-slanting hash
marks); blue horizontal bars, alpha-like globin gene expression; black horizontal
bars, beta-like globin expression; yellow area, yolk sac hematopoiesis;
green area, transition from yolk sac to liver; blue area, liver hematopoiesis;
purple area, transition from liver to bone marrow; red area, bone marrow
hematopoiesis. taken from Lensch
and Daley, Origins of mammalian hematopoiesis: in vivo paradigms and in
vitro models. Curr Top Dev Biol. 2004;60:127-96. |
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The
process of hematopoietic cell formation from ES cells as well as the scale
of the intermediates generated therein. (A) A mouse (line CCE) embryoid
body at day 14 of culture. Several prominent blood islands may be noted.
EB's are then disrupted and replated in coculture with stromal cell underlayers.
Following coculture, hematopoietic precursors are replated into methylcellulose
that contains hematopoietic growth factors in order to determine (retrospectively)
the progenitor content of the input material. (B) A typical day 10 colony-forming
unit granulocyte, erythrocyte, macrophage, and megakaryocyte (CFU-GEMM).
(C) A composite of representative myeloid cell types found in CFU-GEMM following
staining with W/G. taken
from Lensch and Daley, Origins of mammalian hematopoiesis: in vivo paradigms
and in vitro models. Curr Top Dev Biol. 2004;60:127-96. |
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Effect
of Doxycycline Induction of HoxB4 on EB Cells in vitro (A) Colony
formation by cells from day 6 EB controls, and day 6 EBs treated with doxycycline
from day 4 to day 6, in hematopoietic methylcellulose suspension culture.
HPP-GEMM refers to dense GEMMs not normally seen at day 6 of EB development.
(B) Morphology of a typical day 6 GEMM. (C) Morphology of a doxycycline-induced
HPP-GEMM at the same magnification as in (B). (D) A colony of semiadherent
cells from HoxB4-induced day 6 EB cells plated on OP9 in the presence of
doxycycline. (E) Cytospin preparation of these cells growing on OP9. (F)
Colony-forming activity of doxycycline-induced cells grown on OP9 in methylcellulose
with myeloid cytokines. Control indicates doxycycline not added to methylcellulose
cultures; +dox indicates doxycycline induction maintained during methylcellulose
culture. (G) Cytospin preparation from a GEMM obtained from cultures described
in (F). Open arrowheads show erythroblasts; filled arrowhead shows megakaryocyte.
taken from Kyba M, Perlingeiro RC,
Daley GQ. HoxB4 confers definitive lymphoid-myeloid engraftment potential
on embryonic stem cell and yolk sac hematopoietic progenitors. Cell. 2002
Apr 5;109(1):29-37. |
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Fig.
5. Clonal analysis of hematopoietic populations of mice engrafted with ESC-derived
HSCs, as determined by Southern hybridization analysis of retroviral integration
sites. (A) Structure of the retroviral vector MSCV-HoxB4-ires-GFP. Probes
used in Southern hybridization analysis are indicated. (B Left) Southern
analysis of fractionated myeloid and lymphoid populations from primary (1ry)
and unrelated secondary (2ry) engrafted mice, showing multiple comigrating
fragments. (B Right) Bone marrow and spleen cells from two primary engrafted
animals and comparable tissue from the corresponding secondary animals,
showing comigrating fragments. (C) Southern analysis of hematopoietic tissues
from one primary and two corresponding secondary recipients engrafted with
ESC-HSCs: spleen (S), BM (B), Gr1+ BM cells (B/G), Gr1+ splenocytes (S/G),
and CD3+ or B220+ splenic lymphocytes (S/L). Mye/Lym represents the ratio
of Gr-1+ cells to CD3+ and B220+ populations in corresponding sample, as
determined by flow cytometry. Relative DNA level was calculated by comparing
endogenous HoxB4 (endog) with control (DNA isolated from Ainv15 ES cells).
Proviral copy number was calculated by comparing the level of proviral HoxB4
(Rv-HoxB4) with endogenous HoxB4 level. Samples reflect Cdx4/HoxB4-engrafted
cells, except the third and fourth lanes in B Left, which represent HoxB4-treated
cells. #, fragments detected only in primary recipients; *, fragments unique
to secondary engrafted animals; ^, fragments detected predominantly in one
lineage. taken from Wang, Yates, Naveiras, Ernst, and Daley.
Embryonic stem cell-derived hematopoietic stem cells. PNAS December 27,
2005 vol. 102 no. 52 19081-19086. go
to abstract online |
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