Blood, 1 July 2001, Vol. 98, No. 1, pp. 3-5
FOCUS ON HEMATOLOGY
Introduction: spatial origin of murine hematopoietic
stem cells
Mervin C. Yoder
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Article |
Hematopoiesis is the developmentally regulated and
tissue-specific process of blood cell production. The predominant
anatomic site of hematopoiesis changes several times during murine and human ontogeny. Blood and endothelial cells become morphologically identifiable on embryonic day 7.5 (E7.5) in the developing murine yolk
sac blood islands.1 Upon initiation of blood flow through the systemic circulation, blood island-derived progenitor cells are
carried throughout the yolk sac and embryo.2,3 The liver becomes colonized with hematopoietic progenitor and stem cells at the
28 somite pair (sp) stage of murine development.4,5 By E12
the liver is the predominant site of hematopoiesis. Hematopoietic stem
cells (HSCs) in the rodent fetal liver subsequently migrate to the bone
marrow and contribute to lifelong hematopoiesis.6
For nearly 100 years, hematologists have been challenged by the
question of how hematopoiesis is sequentially initiated in different
organs during mammalian development. At the turn of the century,
morphologic evidence that all blood cells appeared to develop from a
common precursor cell was accumulating. The "mother" stem
cell was identifiable as a mononuclear cell with a large nucleus,
prominent nucleoli and deeply basophilic staining cytoplasm.7-9 This cell was present in every tissue
displaying hematopoietic activity, leading Maximow8 and
Danchakoff7 to hypothesize that the stem cell must arise
independently in each hematopoietic tissue at a specific time or under
specific circumstances. Seventy-five years later, the fetal liver has
clearly been established as the site of development for HSCs that seed the bone marrow compartment;6,10 however, the temporal and spatial origin of the HSCs that seed the fetal liver remains
controversial.11,12
More than 30 years ago, Moore and Owen13 hypothesized that
the sequential emergence of hematopoietic organs throughout ontogeny required an inflow of circulating HSCs from the bloodstream and that
HSCs are first formed in the yolk sac. The results of transplantation experiments in which yolk sac cells were injected into irradiated chick
embryos14 served as the basis for this hypothesis.
Follow-up studies in the murine system confirmed that the yolk sac was
critical to the establishment of hepatic hematopoiesis. Removal of the fetal liver before the 28 sp stage (E9.5) and grafting of the tissue
beneath the kidney capsule of an adult recipient resulted in survival
of the fetal hepatic tissue, but no hematopoietic elements were
present. But administration of hematopoietic cells into the circulation
of the recipient mice bearing the fetal tissue grafts resulted in
multilineage engraftment in the implanted fetal liver
tissue.4,5 These data suggested that hematopoiesis did not
arise from precursors endogenous to the fetal liver, but the liver
tissue did promote growth and differentiation of circulating precursors
that lodged therein. Likewise, E7 embryos dissected free from yolk sac
membranes grew and developed normally in vitro for 2 days, but no blood
cells or hematopoietic colony-forming cells were present in any portion
of the embryo, including the liver.15 These studies
supported the theory of organ seeding by blood-borne stem cells and the
yolk sac as the source of HSCs.13
In contrast, some studies in both avian and murine systems have
provided data that refutes the hypothesis of the yolk sac as the site
of origin of HSCs. Using chick-quail and chick-chick chimeric models, a
number of investigators failed to observe yolk sac cell contributions
to adult myleopoiesis or lymphopoiesis (reviewed in Dieterlen-Lievre
and Le Douarin16 and Dieterlen-Lievre et al17).
The results of these avian studies contrasted with those of Moore and
Owen,14 but the experimental methods differed in age of
donor embryonic tissue, model (unirradiated chimeric host versus
irradiated recipient), and period of analysis after transplantation or
chimeric grafting.
Efforts to assay directly for the presence of HSCs in the murine yolk
sac via transplantation of these cells into recipient animals have
yielded results that support or refute the Moore-Owen hypothesis largely dependent upon the age of the recipient. Weissman et
al18 reported that in utero transplantation of E8 yolk sac cells into the yolk sacs of congenic nonablated recipients contributed to lymphomyelopoiesis in surviving mice that grew into adulthood. In
utero transplantation of E9 yolk sac cells into the placenta of
stem-cell deficient congenic fetal mice resulted in long-term reconstitution (more than 5 months) of the erythroid lineage in some
surviving animals.19 Thus HSCs that engraft in embryonic or fetal mice and then contribute to adult hematopoiesis can be observed to be present in the E8-9 murine yolk sac. In contrast, the
first HSCs that directly engraft in lethally irradiated adult mice are
present at E10 in the aorto-gonad-mesonephros (AGM) region within the
embryo.20 Adult-repopulating HSCs cannot be identified in
the yolk sac until E11, at which time HSCs are already present in the
AGM region, the liver, and vitelline and umbilical
vessels.20-23 These results are generally interpreted to
infer that the yolk sac is not a site where fetal-liver-seeding (and
likewise, adult-marrow-repopulating) HSCs develop.
The inability to demonstrate engraftment of yolk sac cells isolated
earlier than E11 in lethally irradiated adult mice may be due to a
failure of the yolk sac HSCs to home and engraft in an adult
microenvironment. Based on the observation that the liver of the
newborn mouse continues as an active hematopoietic organ for 1 to 2 weeks following birth,24-26 sublethally myeloablated newborn mice were transplanted with E9 or E10 yolk sac cells. In
multiple experiments, yolk sac cells were noted to give rise to all
lineages of blood cells long-term (more than 11 months) and that marrow
from the primary E9 and E10 yolk sac-engrafted recipients also
reconstituted B- and T- lymphocyte, granulocyte, and erythroid lineages
in secondary lethally irradiated adult recipient
mice.27-29 These results suggested that yolk sac HSCs capable of repopulating lymphoid and myeloid lineages in adult animals
were present prior to E11 but were not identifiable upon direct
transplantation into adult recipients. What then is the relationship
between yolk sac HSCs that engraft in newborn recipients and contribute
to long-lived multilineage repopulation and HSCs from the AGM region,
the liver, and vitelline and umbilical cord vessels that directly
repopulate all lineages of a lethally irradiated adult mouse?
In this issue, Matsuoka et al30 present novel data that
early (day 8.0 and 8.5 post coitus [pc]) yolk sacs (YSs) and
para-aortic splanchnopleures (P-Sps) possess HSCs that
acquire the capacity to engraft directly and repopulate multiple
hematopoietic lineages in lethally irradiated adult recipient mice only
after a period of coculture with a stromal cell line previously
isolated from the AGM region. AGM-S3 cells are a clonal endothelial
cell line derived from the AGM region of a day 10.5 pc murine embryo.
These cells have previously been demonstrated to support primitive
murine and human hematopoiesis in vitro.31 In the present
work, YSs and P-Sps were isolated from Ly-5.2-staged embryos (stages
not stated), 2 embryo equivalents of cells cocultured with the AGM-S3 cells, and were transplanted (with Ly-5.1-unfractionated adult marrow
competitors) into lethally irradiated Ly-5.1 adult mice. Both day 8.0 and 8.5 YS and P-Sp cells engrafted and repopulated erythroid, myeloid,
and lymphoid lineages but only if cocultured for a minimum of 4 days on
the AGM-S3 cells. As one would predict, AGM-S3 cells alone did not
repopulate hematopoiesis in the recipients.
These data are significant in addressing several questions regarding
the origin of HSCs during embryogenesis. AGM-S3 cells supported the
generation of HSCs and spleen colony-forming unit cells (CFU-Ss) but
not definitive colony-forming cells (CFCs). While both YS and P-Sp
cells produced some CFCs during the first few days of coculture with
the AGM-S3 cells, the CFCs formed consisted of some macrophage colonies
and erythroid colonies with cells expressing both embryonic and adult
hemoglobins reflective of primitive erythropoiesis. In fact, most CFC
activity declined in the cocultures at the time that the CFU-S and HSC
activity appeared. Thus AGM-S3 cells provide factors that promote
generation of CFU-Ss and HSCs but not CFCs. This result is consistent
with observations in normal embryos that, while the AGM region does not
support the differentiation of mature blood cells, it normally supports
the generation of HSCs and some committed definitive progenitor
cells.32,33
The finding that CFU-Ss and HSCs simultaneously emerge from the
cocultured day 8.0 pc YS and P-Sp tissue is intriguing. The stem cell
theory of hematopoiesis purports that the full hierarchy of
hematopoietic progenitor and mature cells arises from a single stem
cell. Are the day 8.0 pc yolk sac- and P-Sp-derived CFU-S descendents
of the HSCs present in these tissues? Several studies have determined
that multipotent and committed progenitor cells can be directly assayed
from YSs and P-Sps at the E8.25 stage (1-8 sp) of
development.2,3,34,35 Are these hematopoietic progenitor
cells also progeny of the CFU-Ss and HSCs identified in the cocultured
day 8.0 pc YS and P-Sp? If so, the cell cycle kinetics and
differentiation programs must be vastly accelerated compared to the
profile of murine HSC commitment and differentiation observed in the
fetal liver and adult marrow microenvironments. An alternative proposal
suggests that the embryonic hematopoietic hierarchy emerges in the
reverse orientation to the classical adult pathway.36 The
hypothesis states the order of appearance of embryonic hematopoietic
cells begins with CFCs, CFU-Ss, and finally HSCs, and these cells may
have a direct lineage relationship or be derived
independently.36 Isolation of the cells displaying the
CFU-S and HSC activity in the day 8.0 pc yolk sac and P-Sp and use of
stepwise addition of hematopoietic growth factors and/or other stromal
elements to the AGM-S3 cocultures may permit examination of the
complete hematopoietic hierarchy in vitro.
An important consideration in the design of the work of Matsuoka et al
was to isolate YS and P-Sp cells from embryos at a time prior to
establishment of the systemic circulation. The exact time of onset of
the circulation has never been definitively established. Cumano et
al37 and Kaufman38 state that the vascular
connection between yolk sac and embryo is established at the 8-10 sp
stage. Rugh39 notes that the heart is beating regularly and
the circulation is playing a major role in development by the 13 sp
stage (day 9 pc). Therefore, while it is not absolutely clear when
forward flow of blood through the yolk sac and embryonic vasculature is accomplished, isolation of the day 8.0 pc YS and P-Sp tissue to examine
for endogenous HSC emergence is consistent with our current level of
understanding that these tissues were not exchanging cells via the circulation.
The simultaneous autonomous emergence of HSC activity in day 8.0 pc YSs
and P-Sps is of great interest. Blood cells in the yolk sac
differentiate from extraembryonic mesoderm derived from the posterior
of the primitive streak.40 Blood cells in the P-Sp are
derived from the splanchnopleure comprising ventral lateral plate
mesoderm apposed to endoderm.41 What are the specific inductive events that commit mesoderm in 2 vastly different tissue microenvironments to simultaneously produce cells with HSC activity? Many of the key molecules involved in the induction of the blood cell
program have been identified (reviewed in Zon,42
Shivdasani and Orkin,43 and Belaussoff et
al44). Interpretation of the present data that the AGM-S3
cells somehow "educate" existing embryonic HSCs to display
competence to engraft in the adult conditioned recipient presents a new
complex area for investigation. Of interest, Cai et al45
recently reported that the level of expression of the Runx 1 transcription factor influences the temporal and spatial appearance of
adult repopulating HSCs. Expression of the 
1 integrin receptor is
necessary for P-Sp cells to home and engraft in fetal liver and the
adult marrow cavity.46 These 2 examples suggest that a
wide variety of changes in cell function may be occurring in the YS and
P-Sp cells in the presence of the AGM-S3 cells.
While day 8.0 pc YS and P-Sp cells have the capacity to engraft and
repopulate the hematopoietic system in conditioned adult hosts if
"educated" by AGM-S3 cells, one may question whether either site
normally contributes the HSCs that seed the fetal liver and establish
the HSCs for lifelong hematopoiesis. That question remains to be
addressed. Additional questions include the following: (1) What is the
frequency of these HSCs in the day 8.0 pc YS and P-Sp and are there
differences in repopulating ability between HSCs emerging in the yolk
sac and those in the P-Sp? (2) What is the relationship between the
HSCs generated in vitro in coculture with the AGM-S3 cells and the
embryonic HSCs that repopulate conditioned newborn recipients? (3) Are
there other tissues, such as the fetal liver, that may comprise
endothelial cells that also possess the capacity to induce adult
engrafting properties from day 8.0 pc YSs and P-Sps?
Perhaps the most significant outcome of the studies of Matsuoka et al
is the demonstration of murine HSC induction ex vivo. At present, it is
unclear if the YS and P-Sp cells that engrafted the conditioned adult
recipients were cells already committed to a hematopoietic fate (an
embryonic HSC) or if these HSCs generated in vitro were derived from a
more primitive cell progenitor (hemangioblast or even residual
mesoderm) or another cell type (endothelium). The increasing awareness
that HSCs display a high degree of plasticity provides new challenges
to define experimental conditions that will permit stem cell induction
and commitment to specific cellular lineages. The model presented by
Matsuoka et al suggest that HSCs may be modified by microenvironmental
manipulation to display new properties and functions but with retained
in vivo multipotency.
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References |
1.
Haar J, Ackerman G.
A phase and electron microscopic study of vasculogenesis and erythropoiesis in the yolk sac of the mouse.
Anat Rec.
1971;170:199-223[CrossRef][Medline]
[Order article via Infotrieve].
2.
Palis J, Robertson S, Kennedy M, Wall C, Keller G.
Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.
Dev.
1999;126:5073-5081[Abstract].
3.
Palis J, Chan R, Koniski A, Patel R, Starr M, Yoder M.
Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis.
Proc Natl Acad Sci U S A.
2001;98:4528-4533[Abstract/Free Full Text].
4.
Johnson G, Moore M.
Role of stem cell migration in initiation of mouse fetal liver hematopoiesis.
Nature.
1975;258:726-728[CrossRef][Medline]
[Order article via Infotrieve].
5.
Houssaint E.
Differentiation of the mouse hepatic primordium, II: extrinsic origin of the haemopoietic cell line.
Cell Diff.
1981;10:243-247[CrossRef][Medline]
[Order article via Infotrieve].
6.
Clapp D, Freie B, Lee W-H, Zhang Y-Y.
Molecular evidence that in situ-transduced fetal liver hematopoietic stem/progenitor cells give rise to medullary hematopoiesis in adult rats.
Blood.
1995;86:2113-2122[Abstract/Free Full Text].
7.
Danchakoff V.
Origin of the blood cells: development of the haematopoietic organs and regeneration of the blood cells from the standpoint of the monophyletic school.
Anat Rec.
1916;10:397-413[CrossRef].
8.
Maximow AA.
Relation of blood cells to connective tissues and endothelium.
Physiol Rev.
1924;4:533-563[Free Full Text].
9.
Sabin FR.
On the origin of the cells of the blood.
Physiol Rev.
1922;2:38-69[Free Full Text].
10.
Zanjani E, Ascensao J, Tavassoli M.
Liver-derived fetal hematopoietic stem cells selectively and preferentially home to the fetal bone marrow.
Blood.
1993;81:399-404[Abstract/Free Full Text].
11. Yoder M, Palis J. Ventral (Yolk Sac) Hematopoiesis in the Mouse. In:
Zon L, ed. Hematopoiesis. London, United Kingdom: Oxford University
Press. In press.
12.
Dzierzak E, Medvinksy A, de Bruijn M.
Qualitative and quantitative aspects of haematopoietic cell development in the mammalian embryo.
Immunol Today.
1998;19:228-236[CrossRef][Medline]
[Order article via Infotrieve].
13. Moore M, Owen J. Stem-cell migration in developing myeloid and lymphoid
systems. Lancet. 1967:658-659.
14.
Moore M, Owen J.
Chromosome marker studies in the irradiated chick embryo.
Nature.
1967;215:1081-1082[CrossRef][Medline]
[Order article via Infotrieve].
15.
Moore M, Metcalf D.
Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo.
Br J Haematol.
1970;18:279-296[Medline]
[Order article via Infotrieve].
16.
Dieterlen-Lievre F, Le Douarin N.
Developmental rules in the hemopoietic and immune systems of birds: how general are they?
Sem Dev Biol.
1993;4:325-332[CrossRef].
17.
Dieterlen-Lievre F, Godin I, Pardanaud L.
Where do hematopoietic stem cells come from?
Int Arch Allergy Immunol.
1997;112:3-8[Medline]
[Order article via Infotrieve].
18.
Weissman I, Papaioannou V, Gardner R.
Fetal hematopoietic origins of the adult hematolymphoid system. In:
Clarkson B,Marks PA,Till JE, eds.
Differentiation of Normal and Neoplastic Hematopoietic Cells. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1978:33-47.
19.
Toles JF, Chui DHK, Belbeck LW, Starr E, Barker JE.
Hemopoietic stem cells in murine embryonic yolk sac and peripheral blood.
Proc Natl Acad Sci U S A.
1989;86:7456-7459[Abstract/Free Full Text].
20.
Muller A, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E.
Development of hematopoietic stem cell activity in the mouse embryo.
Immun.
1994;1:291-301.
21.
Huang H, Auerbach R.
Identification and characterization of hematopoietic stem cells from the yolk sac of the early mouse embryo.
Proc Natl Acad Sci U S A.
1993;90:10110-10114[Abstract/Free Full Text].
22.
Lu L-S, Wang S-J, Auerbach R.
In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietaic precursor cells expanded > 100-fold by coculture with a clonal yolk sac endothelial cell line.
Proc Natl Acad Sci U S A.
1996;93:14782-14787[Abstract/Free Full Text].
23.
de Bruijn M, Speck N, Peeters M, Dzierak E.
Definitive hematopoietic stem cells first develop within major arterial regions of the mouse embryo.
EMBO J.
2000;19:2465-2474[CrossRef][Medline]
[Order article via Infotrieve].
24.
Wolf N, Bertoncello I, Jiang D, Priestley G.
Developmental hematopoiesis from prenatal to young-adult life in the mouse model.
Exp Hematol.
1995;23:142-146[Medline]
[Order article via Infotrieve].
25.
Harrison D, Astle C.
Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: models for human umbilical cord blood.
Blood.
1997;90:174-181[Abstract/Free Full Text].
26.
Ema H, Nakauchi H.
Expansion of hematopoietic stem cells in the developing liver of a mouse embryo.
Blood.
2000;95:2284-2288[Abstract/Free Full Text].
27.
Yoder M, Hiatt K.
Engraftment of embryonic hematopoietic cells in conditioned newborn recipients.
Blood.
1997;89:2176-2183[Abstract/Free Full Text].
28.
Yoder M, Hiatt K, Mukherjee P.
In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.
Proc Natl Acad Sci U S A.
1997;94:6776-6780[Abstract/Free Full Text].
29.
Yoder MC, Hiatt K, Dutt P, Mukherjee P, Bodine DM, Orlic D.
Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac.
Immun.
1997;7:335-344.
30.
Matsuoka S, Tsuji K, Hisakawa H, et al.
Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleura by aorta-gonad-mesonephros region-derived stromal cells.
Blood.
2001;98:6-12[Abstract/Free Full Text].
31.
Xu M, Tsuji K, Ueda T.
Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines.
Blood.
1998;92:2032-2040[Abstract/Free Full Text].
32.
Medvinsky A, Dzierzak E.
Definitive hematopoiesis is autonomously initiated by the AGM region.
Cell.
1996;86:897-906[CrossRef][Medline]
[Order article via Infotrieve].
33.
Godin I, Dieterlen-Lievre F, Cumano A.
Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus.
Proc Natl Acad Sci U S A.
1995;92:773-777[Abstract/Free Full Text].
34.
Rich I.
The developmental biology of hemopoiesis: effect of growth factors on the colony formation by embryonic cells.
Exp Hematol.
1992;20:368-370[Medline]
[Order article via Infotrieve].
35.
Wong P, Chung S, Chui D.
Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac.
Proc Natl Acad Sci U S A.
1986;83:3851-3854[Abstract/Free Full Text].
36.
Dzierzak E, Medvinsky A.
Mouse embryonic hematopoiesis.
Trend Genet.
1995;11:359-366[CrossRef][Medline]
[Order article via Infotrieve].
37.
Cumano A, Dieterlen-Lievre F, Godin I.
Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura.
Cell.
1996;86:907-916[CrossRef][Medline]
[Order article via Infotrieve].
38.
Kaufman MH.
The Atlas of Mouse Development. London United Kingdom: Academic Press Limited; 1992.
39.
Rugh R.
The Mouse. New York, NY: Oxford University Press; 1993.
40.
Lawson K, Meneses J, Pedersen R.
Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.
Development.
1991;113:891-911[Abstract].
41.
Lawson KA, Hage WJ.
Clonal analysis of the origin of primordial germ cells in the mouse.
Ciba Fond Symp.
1994;182:68-91.
42.
Zon L.
Developmental biology of hematopoiesis.
Blood.
1995;86:2876-2891[Abstract/Free Full Text].
43.
Shivdasani R, Orkin S.
The transcriptional control of hematopoiesis.
Blood.
1996;87:4025-4039[Free Full Text].
44.
Belaussoff M, Farrington S, Baron M.
Hematopoietic induction and respecification of A-P identity by visceral endoderm signaling in the embryo.
Development.
1998;125:5009-5018[Abstract].
45.
Cai Z, de Bruijn M, Ma X, et al.
Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo.
Immun.
2000;13:423-431.
46.
Potocnik A, Brakebusch C, Fassler R.
Fetal and adult hematopoietic stem cells require 1 integrin function for colonizing fetal liver, spleen, and bone marrow.
Immun.
2000;12:653-663.
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