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17 JANUARY 2013 I VOLUME 121, NUMBER 3
● ● ● HEMATOPOIESIS & STEM CELLS
Comment on Nakajima-Takagi et al, page 447
Hemogenic
endothelium in a dish
---------------------------------------------------------------------------------------------------------------Michael Kyba1
1 LILLEHEI
HEART INSTITUTE AND DEPARTMENT OF PEDIATRICS, UNIVERSITY OF MINNESOTA
In this issue of Blood, Nakajima-Takagi et al use human embryonic stem (ES) and
induced pluripotent stem (iPS) cells to identify Sox17 as a regulator of hemogenic
endothelium, and use conditional expression of Sox17 to capture endothelial cells
at the critical moment of hemogeneic transition.1
Sox17-overexpressing hemogenic endothelial cells. iPS-derived endothelial cells or pre-HPCs transduced with
Sox17 were locked into an adherent, proliferative state, and expressed both endothelial and hematopoietic
markers. See Figure 2B in the article by Nakajima-Takagi et al that begins on page 447.
hat hematopoietic stem cells derive from
specialized endothelial cells during embryogenesis is now well accepted, thanks to
elegant early studies in avian systems,2,3 studies that have recently been backed up with
direct observation in mouse and fish embryos.4,5 However, much remains mysterious
about the endothelial origins of hematopoietic
stem cells, not least the question of why the
process needs to happen in this way at all.
Only 2 components of the molecular mechanism have been investigated deeply to date,
T
both in the mouse system: Runx1, a positive
regulator, necessary for the hemogenic transition,6 and HoxA3, a negative regulator that
forces hemogenic endothelial cells to stay
endothelial.7 Understanding the mechanism
by which embryonic endothelial cells undergo this hemogenic transition will provide
important insight into the making of hematopoietic stem cells, and may even lead to
that Holy Grail, the generation of repopulating hematopoietic stem cells in vitro from
iPS cells.
BLOOD, 17 JANUARY 2013 䡠 VOLUME 121, NUMBER 3
To this end, Nakajima-Takagi et al developed an improved culture system for the differentiation of human iPS cells allowing them
to efficiently generate cells at different stages
of hemogenesis: endothelial cells (defined as
CD34⫹ CD43⫺ CD45⫺), prehematopoietic
progenitor cells (pre-HPCs), which have acquired CD43 expression,8 and HPCs, that
have further acquired CD45. They then performed a functional screen of a panel of important regulators of early hematopoiesis by
transducing sorted iPS-derived endothelial
cells or pre-HPCs with different retroviruses,
each encoding a different regulator, and following the growth and differentiation of these
cells under the influence of the regulator in
question. Remarkably, whereas genes like
SCL, GATA2, and RUNX1 had no detectable
effect of the further differentiation of these
cells, they discovered that expression of
1 gene, Sox17, drove the expansion of an adherent cell type that expressed both endothelial and hematopoietic markers (see figure). To
better characterize the activity of Sox17,
Nakajima-Takagi et al then modified it by fusion to the estrogen receptor, to regulate its
nuclear localization, and thus activity, by application of 4-OH-tamoxifen to the culture
medium. This allowed Sox17 to be turned on,
and then turned off at a later time point. When
Sox17 was removed, these cells differentiated
into hematopoietic progeny and downregulated endothelial markers, demonstrating
that Sox17 had locked them into a state intermediate between endothelial and hematopoietic.
The investigators then used these captured
hemogenic endothelial cells to investigate the
molecular mechanism underlying the activity
of Sox17. Transcriptional profiling of the
Sox17-induced population revealed that with
regard to transcription factor expression, or
general hematopoietic gene expression, the
Sox17-induced population was more similar to
pre-HPCs and HPCs than to endothelial cells.
ChIP-chip analysis showed that overexpressed
Sox17 bound to about 100 promoters and
417
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regulatory regions (about 1% of those surveyed), including a large number of hematopoietic transcription factor genes, but also
several endothelial-specific genes, such as VEcadherin. Thirty-six genes were in both datasets, that is, both direct targets of Sox17 and
transcriptionally up-regulated.
It seems likely from the transcriptional
profile and ChIP data that Sox17 is initiating a
hematopoietic program while also maintaining
the endothelial program, thus allowing expansion of a cell type stuck between 2 fates. This is
distinct from the 2 previously studied regulators of endothelial hemogenesis: HoxA3, the
negative regulator, promotes the endothelial
program while preventing acquisition of the
hematopoietic program; Runx1 on the other
hand appears to erase the endothelial program
while inducing the hematopoietic program.
It is interesting that several of the genes
targeted and induced by Sox17 expression
were actually screened in the initial experiment, and found to have no effect on proliferation of endothelial or pre-HPC cells. It may be
that combinatorial effects explain the activity
of Sox17. However, the genes in this initial
panel were all selected for playing important
or essential roles in early HPCs. Because hematopoietic differentiation occurs efficiently
without intervention in this iPS culture system, pro-hematopoietic factors might not
present a strong phenotype in this assay. On
the other hand, anti-hematopoietic or proendothelial factors would be predicted to have
clear phenotypes, the latter possibly explaining why Sox17 was found.
It is notable that in addition to a dual
hematopoietic-endothelial phenotype, high
levels of Sox17 expression drive the proliferation of this intermediate cell type. In theory at
least, proliferation is not necessary for hemogenesis. Nakajima-Takagi et al show that under normal circumstances, high levels of
Sox17 persist for only a short time, and in the
earliest (endothelial) fraction, presumably at
the moment of their hemogenic commitment.
High levels of Sox17 thus appear to “kickstart” hemogenesis. Whether the concomitant
proliferation might serve some biophysical
purpose, facilitating bulging of cells out of the
epithelial sheet for example, remains to be
determined. In any event, the sustained proliferation of this transitional population by
Sox17 is serendipitous in that it allows the
generation of large numbers of these cells for
study, which should facilitate a number of
418
important experiments. For example, as the
mechanism of hemogenesis is still very much a
black box, it will be valuable to determine
which of the 36 directly up-regulated targets
are actually necessary for effects of Sox17 on
differentiation and proliferation. It would also
be interesting to study the cell biologic
changes that occur as hemogenic endothelial
cells convert into hematopoietic cells in a large
scale culture synchronized en masse by Sox17
release. Finally, and most importantly, has
Sox17 brought us any closer to the important
goal of developing transplantable hematopoietic stem cells from human iPS cells in vitro?
Having embryonic origins in a sustained hemogenic endothelium is a feature that distinguishes intra-embryonic definitive repopulating hematopoietic stem cells from their earlier
yolk sac nonrepopulating cousins. It will
therefore be very interesting to learn whether
these Sox17-induced human iPS-derived hematopoietic progenitors have in vivo repopulating potential.
Conflict of interest disclosure: The author
declares no competing financial interests. ■
REFERENCES
1. Nakajima-Takagi Y, Osawa M, Oshima M, et al. Role of
SOX17 in hematopoietic development from human embryonic stem cells. Blood. 2013;121(3):447-458.
2. Jaffredo T, Gautier R, Eichmann A, Dieterlen-Lievre F.
Intraaortic hemopoietic cells are derived from endothelial
cells during ontogeny. Development. 1998;125(22):45754583.
3. Sabin FR. Studies on the origin of blood vessels and of
red blood corpuscles as seen in the living blastoderm of
chicks during the second day of incubation. Contrib Embryol. 1920;9:213-262.
4. Bertrand JY, Chi NC, Santoso B, Teng S, Stainier DY,
Traver D. Haematopoietic stem cells derive directly from
aortic endothelium during development. Nature. 2010;
464(7285):108-111.
5. Boisset JC, van Cappellen W, Andrieu-Soler C, Galjart N,
Dzierzak E, Robin C. In vivo imaging of haematopoietic
cells emerging from the mouse aortic endothelium. Nature.
2010;464(7285):116-120.
6. Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E,
Speck NA. Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature. 2009;
457(7231):887-891.
7. Iacovino M, Chong D, Szatmari I, et al. HoxA3 is an
apical regulator of haemogenic endothelium. Nat Cell Biol.
2011;13(1):72-78.
8. Vodyanik MA, Thomson JA, Slukvin II. Leukosialin
(CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures. Blood. 2006;
108(6):2095-2105.
● ● ● LYMPHOID NEOPLASIA
Comment on Richter et al, page 423
Can NKT cells extinguish
smoldering
myeloma?
---------------------------------------------------------------------------------------------------------------Don M. Benson Jr1
1 THE
OHIO STATE UNIVERSITY COMPREHENSIVE CANCER CENTER
One of the greatest challenges in all of medicine is to improve on the life of an
asymptomatic patient; however, in this issue of Blood, Richter and colleagues share
provocative new data taking steps toward accomplishing this goal.1
n this study, ␣-galactosylceramide (␣GalCer)–loaded dendritic cells (DC) were
used in combination with lenalidomide to
elicit multi-component, immune activation in
patients with asymptomatic myeloma.1 Treatment appeared to be well tolerated and was
associated with effects in several immune cell
subsets. A reduction in M-protein was observed in 3 of 4 patients with measurable disease. This innovative approach builds nicely
on prior studies and makes severalimportant,
new contributions to ongoing efforts toward
effective immunotherapy of myeloma.
Multiple myeloma proceeds from a “premalignant” phase termed “monoclonal gammopathy of uncertain significance” (MGUS)
I
through an asymptomatic “smoldering” phase
to active, clinically symptomatic disease.2
While the course of MGUS is variable, most
patients with asymptomatic myeloma will
inexorably progress to manifest signs and
symptoms requiring therapy and ultimately
die of their disease. Although a topic of ongoing investigation, the standard of care for
patients with asymptomatic myeloma remains vigilant observation and withholding
therapy until signs or symptoms of active
disease appear.2 Development of clinical
myeloma from asymptomatic precursor
states is associated with worsening immune
dysfunction, generating increasing interest
in the opportunity for early intervention
BLOOD, 17 JANUARY 2013 䡠 VOLUME 121, NUMBER 3
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2013 121: 417-418
doi:10.1182/blood-2012-12-469940
Hemogenic endothelium in a dish
Michael Kyba
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