Note: Descriptions are shown in the official language in which they were submitted.
WO93/~2182 2 ~ 1 3 ;~ ~ ~ PCT/~S92/0591~
YOLK SAC STEM CELLS
l. INTRODUCTION
The present invention is directed to yolk
sac stem cell In particular, it relates to the
characterization, culturing, and uses of yolk sac stem
cell~ ~or hematopoietic recon~titution and therapy.
Yolk sac st~m cells isolated from the early embryonic
yolk sa prior to blood island formation exhibit a
homogeneous morphology and a primitive cell surface
phenotype without the expression of mature leukocyte
markers and major histocompatibility complex encoded
antigens. The cells can be cultured and expanded
long-term without alteration of their pluripotency.
lS Therefore, yolk sac stem cells may have a wide range -~
of applications including but not limited to the
: reconstlt~tion of a destroyed or deficient human
: hematopoietic system, and the construction of large
and smalI animal models for the production of human
2 0 blood cells, human antibsdies, and testing of human
: disPases~ immune function, Yaccines~ drugs and
;~ immunotherapy. ~ :
2. BACKGROUND~OF THE INVENTION
~ : ~ multip~ten~ial stem cell population is
capable~of giving~ riee to blood cells of diverse
: :morphology and function ~Golde, l99l, Scientific
: ~ .
E American,~December:~6). Since blood cell formation is
first detectable ~in~the~:embryonic yolk sac Parly in
30~ embryogen~sis, it has been hypothesized that
pluripotent hematopoietic stem cells may be present
within the yolk sac, but the charact~ristics of such
cells are still:poorly understood and such cells have
not hereto~ore been identified (Moore and ~etcalf,
: 35~ l970, 18:279). Durin~ fetal dev~lopment, the stem
'
:.
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WO93/02182 2 ~ 1 3 i 5 5 - 2 - PCT/US92/05918
cells migrate to the fetal liver where they reside
temporarily, and eventually move to give rise to the
bone marrow which is the permanent site of blood cell
formation in the adult. Studies on the development of
blood cells have led to the identification of a
variety of important growth and differentiation
factors that regulate hematopoissis. Further, tissue
typing technology has ushered in dramatic advances in
the use of hematopoietic stem cells as a form of
therapy in patients with deficient or abnormal
- hematopoiesis.
2.l. HEMATOPOIETIC STEM CELLS
A pluripotent stem cell is believed to be
lS capa~le of self-renewal and differentiation into blood
cells of various lineages inc7uding lymphocytes,
granulocytes, macrophages/monocytas, erythrocytes and
~ - megakaryocyte (Ikuta et al., lg92, AnnO Rev. Immunol.
:~ 10:759). The mechanism by which a stem cell commits
:~ 20 to a specific cell lineage has not been fully
elucidated. The mech~nisms involv~d in stem cell
replication without differen~iation are also unknown.
; However, it is Glear that such eYents must, in part,
:be influenced by a variety of growth and
25 :~differentiation fac~ors that specifically regulate
~ hema~opoiesis. Other factors which are not yet
:; ~ : identified may also be involved (Me~calf, 1~89, Nature
E : 339:27~. The :commonly known hematopoietic factors
include :erythropoietin (EPO), granulocyte~macrophage
30 colony stimulating factor (G/M-C5F), granulocyte
colony-~timulating factor (G-CSF), macrophage
:colony-stimulating ~M-CSF), interleukin l-l~ (IL-l to
IL-12~, and stem cell factor (5CF~.
An~understanding of hematopoie~is is
: 35 critical to the therapy of h~matopoietic disorders.
~ESTiTIi~L ~ ET
W~93/02182 - 3 - ~ ~ 1 3 ~ ~ t ~ PCT/US92/05918
Neoplastic transformation, immunodeficiency, genetic
abnormalities, and even viral infections all can
affect blood cells of different lineages and at
different stages of development. For ex~mple, basic
knowledge of blood cell development has contributed to
the success of bone marrow transplantation in the
treatment of certain ~orms of hematopoietic
malignancies and anemi~s.
ConYantional therapy utilizes whole bone
marrow harvested from the iliac crest but this
approach has certain limitations. ~one marrow stem
cells are present at extremely low concentrations, and
.they may not be at the earliest stage of
differentiation. An impediment in bone marrow
transplantation is the need for matching the major
histocompatibility complex ~C) between donors and
: recipi~nts through H~A tissue typing techniques~
~ Matching at major loci within the MHC class I and
: ¢las~ II genes is critical to ~he prevention of
rejection responses by the recipi~nt against the
engrafted cells, and more importantly, donor cells may
also mediate an immunological reaction to the host
: tissues referred to as graft versus host disea e. In
ordPr to facilitate graft acceptance by the host,
2S immunosuppr~ssive agents often have been employed,
~:~ which render the patients susceptible to a wide range
of opportunistic infections.
Hollands~examined the in vivo potential of
embryonic cells, and ~ound that day 7 embryonic mou~e
cells could colonize the hematopoietic system of
normal non-irradiated allogeneic mice (Hollands, 1988,
British J. Haematol. 69:437). However, it was not
clear which embr~o~ic cell population actually
contributed to thi~ result, a~ total ambryonic cells
were used for in vivo transfsr. In a study on the
.
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~ ~ 1 3 . j ~ j
effects Qf ~n utero cell transfer, d~y 9 yolk sac
cells were injected into syngeneic fetuses which
differed from the donor cells only at the ~-globin
1QCUS (Toles et al., 1989, Proc. N~tl. Acad. Sci.
U.S.A. 8~:7456). The donor cells were shown to induce
hematopoiesis. Both of these ln ViYo studies utilized
freshly isolated cells from mouse embryos, and there
was no suggestion that long-term cultured and expanded
embryonic yolk sac cells could retain their
pluripot~ncy. Such methods involved diffusion
chambers ~mbedded within the species o~ origin of the
yolk sac tissue (Symann et al., Exp. Hemat. 6:749,
.1978~ or method~ that led to ln vitro malignant
transformation of the yolk sac cells. For example,
long-term yolk sac cell lines were es~ablished from
day 10-13 mouse embryos, and they were shown to give
rise to tumor cells at high frequency ~Globerson et
: al., 1987, Differentiation 36:~85). Therefore, the
potential of tumor formation renders such long term
cultured cells undesirable for use in reconstitution
~: :
~: therapy.
2.2. N~ ISTOCOMPATIBILITY_COMPLEX
: : The MHC is a highly pol~morphic complex of
~:: : 25 genes (Bach and Sachs, 1987, New Eng. J. Med.
317:489). It was first discovered by its ~lose
association with the phensmenon of transplantation
rejection of tis~ue grafts. Subse~uent studies
conclusi~ely demonstrated that antigens encoded by MHC
class~I gene~ are the major targets of transplantation
rejection responses. Such antigens are expressed by
all somatic cells.
MHC class II genes encode molecules on a
limited array of cells, most of ~hich are re~ated to
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WO93/02182 2 1 ~ S~ r 5 r
the hematopoietic system. They can also elicit
reactions by allogeneic immune cells.
Studies on the expression of MHC antigens by
embryonic yolk sac cells yielded inconsistent results.
S Billington and ~enkinson (Transplantation 18:286,
1974), working with cells of the yolk sac of 10-14 day
mouse embryos, found that these cells expres~ed both
H-2 and non-H2 ~murine major and minor
histocompatibility) antigens. The work of Patthey &
Edidîn (Transplantation 15:211, 1973), cited by
Billington and Jenkinson, reported that H-2 antigens
: firs~ appeared on day 7 embryos which could provoke a
.strong immune reaction, but ~he latter suggested that
these antigen~ did not make an appearance in utero
until day 9 or la~er. See THE EARLY DEVELOPMENT OF
~AM~ALS 21g (Balls and Wild, eds., Cambridge U.:1975).
.:~ Heyner reported that H-2 antigens were detectable in
~: day 7 mouse embryos (Heyner, 1973, Transplantation
Ç75). Further, mous~ yolk sac cells obtained at
2~: day 9 of gestation were shown to be capable of
generatin~ a graft-versus-host response in vitro
H~fman and Globerson, ~973, Eur. J. Immunol. 3:1793.
However, Parr~et ~al~ demonstra~ed that H-2 antigens
were absent on the apical or the laterobasal membrane
Z5~of thé mouse yolk~sac~endoderm even at day 20 of
pregnancy (Parr:et~;al., 1980, J. Exp. Med. 152:945).
Thus,:no consensu has been established in regard to
: ;the an~igenicity of yolk sac cells.
'
: 30 3. UMHARY OF THE INVENTION
: The present in~ention relate~ to yolk sac
stem cells, a~method of isolating and culturing yolk
: ~ sac stem cellR ~ and a method of using the cultur~d
yolk sac calls for recsns~ituting an allogen~ic or
x~nogen~ic hematopoietic ~yste~.
'
S~ T~TUTL S~d~E~ ~
WO93/0218? 6 PcT/us92/o5~l8
21 13 ~
. The invention is based, in part, on
Applicants' discovery that the murine yolk sac,
isolated from mouse embryos prior to visible blood
island formation, contains a homogeneous population of
cells that are CD34+, Thy-1 , MHC class I and ~lass
II . Such cells can be e~panded in number by
long-term in vitro culture with minimal
differentiation, and can give rise to mature blood
cell~ of diverse lineages when subsequently treated
0 with the appxopriate hematopoietic growth and
differentiation factors. Further, the long-term
cul~ured cells also can mature into functionally
: ~.competent blood cells ln vivo, capable of mediating
antigen-specific immune responses, repopulating
lympho-hematopoietic organs, and prolonging survival
o~ animal~ with a destroyed hematopoietic system. The
~: yolk sar cells of the invention can be successfully
transplanted into allogeneic ~etuses in utero and into
: non-immunosuppressed xenogeneic hosts ~nd since these
20~ cells do not induce graft-versus-host an~
host-versus graft reactions, tran~plantation will
: ~ ~resuIt:in tissue chimerism.
;~ ~ The i~vention is described by way of
examples in which murine yolk sac cells are isolated,
2~5~ and their cell surface phenotype is characterized.
The h~mogeneous population:of yolk sac cells is
: expanded in long-term culture, and shown to retain
plurip~tency in vitro and in vivo. A wide variety of
ùs~s for the yolk sac cells are encompassed by the
invention described herein.
4. BRTEF DESCRIPTION OF T~E DRAWINGS
FIG. 1. A schematic drawing Qf ~he appearanc~ of
mouse embryos around day 7 and day 8.5 of
gestation.
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W~93J021~2 _ 7 2113 ~ ;~ i PCT/US92/05918
FIG. 2. ~urine yo!k sac cells from a day 7 embryo
are more homogeneous in appearancP than
cells from a day 8.5 embryo by flow
cytometry analysis.
FIG. 3. Murine yolk ~ac cells from a day 7 embryo
express CD34 but not Thy-l, M~C class I and
class II antigens.
FIG, 4. Cultured yolk sac cells can differentiate
into mature blood cells in vitro, including ~;
(4A) monocytes, (4B~ megakaryocytes, (4C)
erythrocytes, and (4D) lymphocytes.
15 FIG, 5. Yolk sac cells recovereà from recipient
mou~e spleens following in YiVC) transfer
demonstrate the expression of mature
laukocyte antigens by donor ce~ls.
FI~. 6. : Hemagglu~ination of red blood cells coated
with antigens (FIG. 6A, lipopolysaccharide,
and FIG . 613, ~ human serum albumin~ by sera of
inununodef icient mice trea l:ed with yolk sac
cells, demonstrating restoration of immune
~: ~ 2s: ~ ~ function by yolk sac ~ells in vivo.
: : :
: FIG.::7. Cultured~yolk sac cells repopulate the
: spleens of chemi~ally-ablated mice and give
~: rise to~colony-forming units in vivo; (7A) A
comparison betw~en a ch~mo-ablated mouse
: ~pleen and a: fully repopulated spleen; (7B~
A repopulated spleen at day 7 post-yolk ac
treatment; (7C) A populat2d ~pleen at day 14
post y~lk-sac treatment.
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WO93/02182 ~ 8 PCT/US92/0S91~
~1 13 ' ~ 5
FIG. .8. In utero injection of yolk sac cells into
allogenic mice leads to tissue chimerism in
new born mice.
FIG. 9. Survival and differentiation of long-term
cultured murine yolk sac cells in a sheep
and a goat which had received multiple high
dos~s of yolk ~ac cells.
105. PETAILED DESCRIPTION OF THE_INVENTION
The present invention relates to yolk sac
stem cells, to methods of isolating and culturing the
.yolk sac stem cells, and to methods of using the yolk
~ac stem cells~
15~lthough the specific procedures and methods
described herein are exemplified using murine yolk sac
cells~ they are merely illustrative for the practice
of the inVention. Analogous procedures and techniques
are equally ap~licable to all mammalian species,
20 iIlcluding human sub~ects. Therefore, human yolk sac
stem cells may be isolated from the embryonic yolk sac
prior to blood island formation. The cell~ having the
phenc)~:ype of CD34~, Thy-l , and MHC class I and II
may be cultured under the same conditions described
25 herein, infra.
Mammalian development may be divided into
three distinct stages: the ~t from fertilization
t~ cleavage; the embryo, from cleava~e to the
formation of all somites; and the fetus, frc3m the
30 formation of the last somite urltil birth~ This
inventiorl takes advantage of the unique properties of
embryonic yolk ~sac cells after their course of
devalop~,lent is dst~rmined, but beiEore l:h~y have los~
either immuno- incomp~tency or the ability to
35 proliferate rapidly.
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W~93~0218~ _ 9 _ PCT/VS92/05918
. It is known that when completely
undifferentiated cells of the blastula or morula are
transplanted into a developed animal, they produce
tumors. These totipotent, tumorigenic cells are of no
S value for in vivo recon5titution ~herapy. However, in
accordance wit~ the invention, it is advantageous to
transplant ~ells which have reached a stage of
specialization at which they have become committed to
a particular sequence of development, or lineage.
Such cells may be used alone or to deliver genetic
material, or it5 expression products, into a
particular tissue of the body, including blood cells.
The cells can be transplanted into a host before or
after transformation with an exogenous gene of
interest, and allowed to de~elop into the tarset
tissue.
While it is necess~ry to use cells which
have matured to the point of losing totipotency, fully
mature cells will be rejected by a histoincompatible
~O host, Consequentlyl it is desirable to use cells
which have just lost totipotency, but still xetain
: pluripotency for a particular tissue typ~. Such cells
: also may retain the ability to colonize, thus
facilitating their delivery to the target tissue.
: 25 Stem cells of the embryonic yolk sac offer
~ particular advantages for hematopoietic
.
re~onstitution. Unlike,the cells of the embryo, the
cells of the~yolk sac develop into only a small number
of dif~erent ~i~sues. Among those ~i sues is the
hematopoietic system, which includes the red and white
blood cells, and the tissue of the vein~, arteries and
capil~aries. Thu~, by day 8 in the development of the
~ use embryo, mesodermal cells in the yolk sac begin
- : to ~orm blood islands. The cells of the blood i~lands
diff~rentiat~, the peripheral cells becoming the
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WO93~02182 l0 PCT/~'S92/05918
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endothelium of the future blood vessels, and the
central cells becoming first mesenchymal cells and
then the red and white blood cells. The blood islands
establish communications to form a circulatory
network, which is extended into the embryo proper.
The yolk sac cells of the subject invention
do not express MHC antigens, and can mature in
allogeneic and xenogeneic hosts, demonstrating their
ability to escape immune rejection. By contrast,
research with bone marrow cells has depended on the
use of immunocompromised hosts. The culture methods
described herein maintain the yolk sac in their
.undifferentiated state, and are applicable to mass
culture of yolk sac cells, providing donor cells for
larqe numbers of recipients.
5.1. ISOLATION OF YOLK SAC CELLS
The embryonic yolk sac is ~he first
dentifiable site of blood cell formation in ontogeny.
The~yolk sac cells travel to the fetal liver during
embryogenesis~and eventually migrate to the bone
marrow~where they~reside~and differentiate into mature
blood cells throughout the entire adult life.
The~embryonic development of the mammalian
2s yolk sac is rapid and occurs within a narrow time
frame. Thé murine yolk sac is fully formed by day 7
of gestation, and~;the formation of blood is detectable
in the mesenchyme~;of the body stalk and in neighboring
;
areas `of the yolk sac. Shortly therea~ter, masses of
mesenchymal cells round up and become aggregated to
form blood islands. By day 8.5, extensive blood
island formation in the murine yolk sac is readily
~isible microscop~ically. At this stage, embryonic
development has~reached a level where fetal liver is
formed and yolk sac cells begin to migrate to the
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W~93/0218~ 3 ~ ~ S PCT/US92/05918
fetal liver. Upon the departure of the yolk sac stem
cells, the yolk sac begins to atrophy. Similar events
also occur in embryonic development of other species,
but the timing of developmental events varies between
S different species. In humans, the yolk sac is formed
by day lQ of gestation, and bl~od island formation
occurs shortly thereafter. Thus, human yolk sac cells
isolated at day lO may be comparable to the murine
cells at day 7.
Since the yolk sac is where blood cell
for~ation is first established in development and the
yolk sac cells eventually reach the bone marrow to
become the bone marrow hematopoietic cells, it is
reasoned that the~yolk s~c represents the earliest
;15 site for the generation of primordial hematopoietic
~- cell precursors~ The cells have committed to the
hematopoietic differentiative pathway so that they are
no longer totipotent.~ However, the yolk sac cells are
still pluripotent,~since~they have not yet committed
20~ to~a~particul~ar blood cel1 lineage as seen by their
ability to make cel1s~of lymphoid, myeloid, and
erythroid~lineages~.~ Hence, yolk sac cells may be the
ideal cell population~for use in reconstitution
therapy including,~but not limited to, bone marrow
25~transplantation~. ~In;;addition, the primitive nature of
these cells, as~evidenced~by the absence of cell
surface expression~of various mature markers and MHC
transplantation~rejection~antigens, may render these --
cells uniquely~capable o~f being used as a universal
~30; donor cell population in allogeneic and even
xenogeneic hosts.~
The isolation of the embryonic yolk sac may
be achieved using~a variety of surgical methods.
Traditionally, the;~yolk sac of a ~ouse embryo is
.-.
~ 35- disaggregated by the use of enzymatic digestion and
:::
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WO93/021g2 - 12 - PCTtUS92/059lX
2 ~
mechanical separation upon surgical removal. A
gentler method of detaching the cells from the yolk
sac membrane and separating them from each other is
described in Section 6.~.1. in which a yolk sac is
immersed in an EDTA solution which causes the cells to
segregate and form a single cell suspension. This
method minimizes cell lysis due to physical force and
cell surface protein alteration due to enzymatic
treatment.
ln Since the establishment of blood islands in
the yolk sac marks the beginning of csllular
differentiation and blood cell ~ormation, it is
preferable that y~lk sac cells be isolated prior to
extensive blood island formation. Large numbers of
highly ho~ogeneous yolk sac cells of day 7 murine
embryos (or similar stage human yolk sac cells), can
be isolated using the method described herein, and
cells obtained at this stage should in principle
: contain the least committed and least differentiated
p~uripotent stem cells suitable for long~term in vitro
culture, or:use in immediate in vivo therapy or as
ca~rriers of specific exogenous genes for use in gene
therapy. : ~
For:long-term maintenance of the yolk sac
Z:S cells, the cells are grown in medium containing a
: relatively high concentration of serum supplement,
between 15-20%. Various cytokines may be added to
: suppress differentiation of the stem cells, including
but not limited to, leukemia inhibitory factor (LIF)
or stem cellifactor/the c-kit ligand (SCF) or SCF in
combination with other cytokines such as IL-3. Such
factors accelerate the multiplication of cultured
~ cells, while inhibiting cellular differentiation in
: : vitro. The examples presented in Section 6, infra,
were ~11 performed using yolk sac cells grown in the
.
~U~T~TLiTL S~ET ~
W~ 93/û2182 -- 13 ~
presence of lO-lOO U/ml of LIF. However, higher LIF
concentrations may be used to achieve stronger
suppression of differentiation. The growth of cells
using SCF could produce similar results.
Alternatively, a number of other known hematopoietic
factors such as I~-3, CSF~s and EPO also may be used
in combination depending on the need to select for a
particular cell type. For example, the combined use
of IL-3 and EPO may assist in driving cultured yolk
sac cells towards the erythroid pathway. The
maintenance of cells at the appropriate temperature,
CO2 concentration, humidity level and the frequency of
changing the culture media are within the ordinary
skill of the art~
5.2. CHARACTE IZATION OF YOLK SAC CELLS
As sh~wn by the examples de~cribed herein,
: yolk sac cells obtained from mous~ embryos prior to
blood island formation are more homogeneous in
~O app~arance ~an ce}ls obtained at a later stage.
Freshly isQlated yolk sac c~lls from day 7 and day 8.5 :.
murine embryos were compared by light scattering using
: : flow cytometry analysis, see Section 6.2.1., infra.
: It is apparent that yolk sac cells of day 7 mouse
embryos are extremely uniform with respect to both
cell size and cell shape. By day 8.5, distinct
populations of cells are clearly visible, suggesting
that the earlier~stage yolk sac cells may be clonally ~:
derived and the difference of 1 day in development may
be cri~ical to the nature of th~ yolk ~ac cells.
Another indication of the primitive nature
: of the early yolk sac cells is their cell surface.
phenotype in regard to the e~pression of various
lineaqe-~ecific blood cell markers. Thi~ form of
analysis may be most conveniently carried out by the
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WO93/02182 - 14 - PCT/US9~/0~91~
2 1 1 ~3 ~'J; ~ J
use of a panel of marker-specific monoclonal
antibodies. When the day 7 yolk sac cells were
reacted with antibodies, the results show~d that they
lacked expression of all mature blood cell markers.
S In addition, such cells did not express MHC-encoded
products which are the major targets of
transplantation rejection responsesO Thus, yolk sac
stem cells can be characterized as CD34~, Thy-l , MHC
class I and MHC class I$ . Similarly, human yolk sac
stem cells obtained from day 10 embryos should display
an îdentical cell suxface phenotype.
The CD34 and Thy-1 markers previously have
been demonstrated to be associated with bone marrow
hematopoietic stem cells (Spangrude et al., 1988,
t5 Science 2~1:58). While CD34 expression declines as
stem cells differentiate and mature, the presence of
Thy~ retained and its density increa~ed in certain .
mature blood cells, particularly T lympho~yt~s. The
finding that yolk sac stem cells are positive for CD34
Z0 expression is consistent with the~e cells being stem
cells. However, the absence of Thy-~ expression
suggests that yolk sac cells may represent an ~arlier
cell popula icn than the bone marrow stem cells which
e~press low levels of Thy-1 in the bone marrow
micro~nviro~ment, In fact, when yolk sac cells ~re
cultured in vitro, a:small percentage of the cells
:~ esrape the effect of LIF and begin to express Thy~
further suggesting that Thy 1 expression is a later
event of stem cell development.
MH~-encoded class I and class II molecules
are involved in immune regulation between T, B, and
antigen presenting cells. ~hese highly polymorphic
molecules also serve as targets in major
transplantation rejection responses between
3S geneti~ally mismatched individuals. Therefore, HL~
.
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Wn93/0~182 2 ~ 5 ~
tissue typing is currently a routine clinical
procedure in ensuring graft acceptance in human
transplant patients by matching the donors and
recipients ~t the major MHC genetic loci. The absence
of MHC antigens on the yolk sac cell surface strongly
suggests the possibility of using such cells as
universal donors in hematopoietic reconstitution
therapy, alleviating the need of tissue typing and the
restrictiv~ use of only MHC-matched tissues as donor
cells. ~he development of adoptively transferred yolk
sac cells in the environment of the host may lead to
specific tolerance between the host and donor cells
~or each other, causing a diminution of the potential
for inducing graft-versus-host and host-versus-graft
lS reactions.
The ~bove-described yolk sac phenotype i5
seen with the va t majority of cells isolated fr~m day
7 murine embryos. Therefore, early isolation of yolk
sac cells pr~vi~es for a highly homogeneous and
: 20 enriched population of stem cells. This is in
; ~ contradistinction to the purification procedure needed
for murine bone marrow hematopoietic stem cells which
are of CD34~ and Thy-1+ phenotype. Such cells must be
isolated and enriched by a sPries of ~election steps,
as they constitute only less than 0.1% of the total
cells in the bone marrow (Spangrude et al., 1991,
Blood 78:1395). On the other hand, yolk sac stem
c~lls can be obtained in an essentially homogeneous
s~ate without requiring additional puri~ica~ion, and
such cells retain their phenotype and functional
act}vity during long-term in itro growth.
..
5.3. FUNCTIONAL ACTIVIT~ES OF Y~OLK SAC CELLS
The pluripotency of yolk sac stem cells to
-~5 differentiate and mature into functionally competent
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WO93/02182 - 16 - PCT/US92/0~9l~
2 ~
blood cells of various hematopoietic lineages was
tested by a number of in vitro and in vivo me~hods
described herein. The presence of a pluripotent
population in long-term cultured yolk sac cells was
5 first demonstrated as follows. After 10 passages of
in itro growth, yolk sac cells were washed from LIF
and exposed to a combination of cytokines including
IL-3, CSF's, and EPO at previQusly determined optimal
concentrations for an additional three weeks in
culture. ~t the end of the period, the stimulated
yolk sac cells were prepared as blood smears and
stained with hematoxylin. The result of this analysis
.reveals the appearance of blood cells that can be
identified as erythrocytes, ~ranulocytes,
megakaryocytes, and l~mphocytes.
~ similar study also was carried out in VlVO
by rec~v~ring donor cells four weeks af~er ia v vo
injection into allogeneic SCID mice. The yolk sac :;
cells used in this study had been expanded in culture
: 20 for o~er 40 passages. Double-staining of the spleen,
bone marrow, and thymus cells of the SCID mice was
performed using antibodi~s ~pecific for the donor cell
haplotype of H-2d and antibodies against mature blood :
: cell markers such as B220 for B cells, CD3 and Thy-1
for T cells, and Mac-l for macrophages~ The results
of this in vivo study confirm the in vitro study that
long-term cultured yolk ~ac cells are capable of
giving rise to mature T cells, B cells and
macrophages/monocytes.
In addition to morphologic evidence of blood
cell maturation f rom yolk sac cells, the adoptively
transferred yolk sac ceIls were tested for functional
activities in the form of specific antibody
production. One month after re~eiYing an infusion of
yolk sac cells, the mice were immunized with either
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W093/02182 17 - 2 1 1 3 ~; ~ P~T/US92/05918
lipopolysaccharide (LPS) or human serum albumin (HSA).
Sera of mice were diluted serially, reacted with the
two antigens, and compared with normal mouse sera as
controls. LPS is a T cell-independent antigen which
activates polyclonal ~ cells directly. The high titer
of LPS specific antibodies in the sera of yolk sac
cell-bearing beige nude xid mice after LPS
immunization indicates the presen~e of functiona~ly
competent antibody producing cells, i.e., B
lympho~ytes and plasma cells. Additionally, HSA,
which is a T cell dependent antigen, elicited a weaker
yet detectable specific antibody production in mice.
Since the a~ti-HSA antibody response requires T cell
help which, in turn, is first activated by antigen-
presenting cells such as macrophages, this resultprovides evidence for the presence of mature and
functional T cells, B c~lls, and macrophag~s which co
operate and interact in the generation of antibodies.
As a corollary, this also suggests that other T cell
20 :and macrophage-mediated functions such as
: cytotoxicity, lymphokine and ~ytokine secretion,
phagocytosis, antigen processing and pres~ntation may
all develop from the transferred yolk ~ac stem cells.
: ~ The ln Yiy~ transfer of yolk sac cells also
~S repopulated ~he spleens of mice whose hematopoietic
system had been previously destroyed by ch~mical
abla-~ion or lethal doses of irradiation. This
resembles situations in which a patient's
lymphohematopoietic system is deficient du~ to a
genetic disorder or an acquired viral in~ection, or a
patient's system is intentionally destroyed by
ch~motherapy or radiotherapy in order to eradicate
tumor cells in the bone marrow. The admini~tration of
- yolk sac cells induc~d colony forming units-sple~n
~U-S) in l~thally irradiated or chemo-ablated mic~
SU~'TITUTE S~E~T
, . . . . . . , . . . .... ...... ..... ....... , ... , .. , .. ..... .. . ~ . .. ... , . ~ .. .. . . .
WO93/02182 - 18 - PCT/~'S92/0591X
2 ~ 1 3 i ri S
whose. spleens, othPrwise, frequently exhibited a
necrotic appearance. On the other hand, expansion of
the yolk sac cells over a period of time in vi~o
supported repopulation and restoration of spleens
S completely normal in appearance. Further, the yolk :
sac cell-treated mice experienced a prolongation of
survival time when compared with the untreated control
group~ Therefor~, long-term cultured yolk sac cells
may be useful in a variety of settings in which bone
marrow reconstitution can be applie~ as an effective
means of therap~
Transplantation of murine yolk sac cells
.into allogeneic fetuses in utero and xenogeneic new
born animal~ did not induce graft rejection reactions.
lS The yolk ~ac cel-s persisted ln vi~o and established
hema~opoietic chimerism in the spleen, li~er, and
pexipheral blood of the host. Thus, yolk sac cell5
may be useful as universal donor cells in various
mammalian species, including humans.
: 5.4. USES OF YOLK SA STEM CELLS
The absence of MHC antigen expreqsion by
~: yolk sac stem cells provides for a source of donor
: cells for in v transplantation and reconstitution
; 25 therapy. The cellB ~ay be used immediately after
isolation from the yolk sac or after long-term
expansion in Yi~ in order to procure larger numbers
for more effective doses. Introduction of ~xogenous
genes into the yolk sac cells may be achi~ved by
conventional method~ during in yitr~ culture and/or in
ivo gene therapy. Long-term cultured c~lls may be
used as a mixed population or progenitors can be
: pre-selected based on the primitive phenotype of
CD34~, Thy-l9 MHC cla~s I and class II , or ~y
limitin~ dilution cloning, prior to in Ya~ use.
TI ~ ~TE S~ET
WO93/021~ - l9 2 ~ ~ 3 J~ 5 PCT~US92/~5918
5.4.l. HUMAN YOLK SAC CELLS IN MICE
Human yolk sac cells may be obtained, grown
in vitro and transferred into immunodeficient or
immunocompromised mice. Such mice contain a human
hematopoietic system and may be used for the study of
human blood cell development in vivo, the
identific~tion of novel hematopoietic growth and
differentiation factors, and testing for cytotoxic
and/or inhibitory compvunds that affect various stages
of blood cell formation as well as anti-cancer drugs.
Such a chimeric mouse referred to as HumatoMo~se~
herein would be superior to the conventional SCID/Hu
mouse model in which mice are reconstituted with human
bone marrow stem cells because HumatoMouse~ wou~d
permit studies in the delineation of the e~rliest
events in hematopoiesis. Furthermore, yolk sac cells
may be implanted in utero in~o nor~al mouse fetuses
~or engraftment of human blood cells in a normal mouse
environment. Such yolk sac cells may be trans~ected
20 with a drug-resistance ~ene so as to allow subsequent
selestive ablation o~ only the host cells using th~
corresponding dru~.
It has been observed that SCID mice are not
totally i~munodeficient and that a small amount of
restoration of immune function is correlated wit~ the
age of he mi~e. SCID mice possess detectable natural
killer cell and macrophage activities. A small
percentage of mice even re-acquire T and ~ cell
function as they mature. Thus, conventiQnal SCID mice
may not be the most appropriate host~ ~or the
construction o~ the HumatoMouse~ as their immune
function may interfere with the analysis of the d~nor
yolk sac cells. The steel mice possess a ~utation at
the steel locus which encodes SCF, a ligand for the
proto-oncogene c-kit cell surf~ce receptor. ~ouse
~IJ~T~F~T~ ~It~
WO93/021X2 PCT/US92/05918
~ ~ 1 3~1~J~` - 20 -
fetuses that are homozygous for this mutation liveonly to about day 15 of gestation before they are
aborted due to the absence of a hematopoietic system
and blood cell formation. Hence, human yolk sac cells
may be injected into the developing homozygous fetuses
in utero prior to abortion, e.g., at day 8, to
reconstitute their hematopoietic function. The
resulting neonates should have a fully humanized
system with no contribution by the host as they would
10~ not normally have lived to birth.
Studies described herein demonstrate that
cultured yolk sac cells can develop into mature blood
.cells in vivo, suggesting that the cells secrete the
necessary growth and differentiation factors for
supporting their own development. A further
i~provement of the Humatomouse~ modPl includes the
introduction o~ human growth and di~ferentiation
factor genes in the mice. In the event that certain
of the~critical cytokines for human blood cell
20 ~formation are species-specific, such as SCF, and mouse
molecules do not~act effectively to promote growth and
differentiation of~human cells, transgenic SCID or
steel~mice may be~constructed to result in endogenous
production of human~cytokines of interest such as
25~ IL-3, CSF's,~and~SCF.~ ~Alternatively, human yolk sac
cells may be transfected with murine receptor genes.
he subsequent transfer of human y~lk sac cells to
5 ~ these~mice should~give rise to a more complete and
efficient human hematopoietic system in mire.
5.4.2. TRANSPLANTATION USING YOLK SAC CELLS
The repeated transfer of high doses of
long-term cultured mouse~yolk sac cells into a foreign
species, i.e. sheep, has shown that the cells persist
~ vivo, differentiate into mature lymphocytes, and do
SU~ i 5TaTL S!~EET
2~ 1~5~
W~93/02182 - 21 - PCT/US92/05918
not mediate graft versus host disease. Although the
mature donor mouse cells eventually express MHC
antigens in vivo, the donor ce~ls are present in high
quantities in the peripheral blood of the xenogeneic
host. The absence of graft rejection (ho~t versus
gra~t) and graft versus host reactions may be
attributed to the primitive nature of the yolk sac
cells, particularly the lack of MHC antigen
expression, allowing the cells and the ho~t immune
~0 system to 'llearn" each other as self prior to MHC
expression and thusl induce a state of specific
tolerance.
Xenogeneic transplants of solid organs have
been carried out in humans in situations where there :~
is a shortage of ~LA-matched organs. With respect to
xenogeneic transplant o~ primitive hematopoietic stem
cells r yolk sac cells may be used to reconstitute the
hematopoietic system of any mammalian ~pecies, for
exa~ple, in a human patient with HIV infection. Since
non-human T cells cannot be infected by human HIV,
: this approach may serve as a means of limiting HIV
- infection in humans. Yolk sac cells may also be
trans~ected with genes which are designed to disrupt
HIV gene sequences involved in HIV replication prior
to in ivo~administration. Such exogenously
: introduced genes may encode anti-sense RNA or ribozyme
: : :molecules that specifically interfere with HIV
replication. Further, the induction of tolerance by
the transfer of xenogsneic yolk sac cells may allow
subsequent transplantation of sol id organs, including
but not limited to heart, liver and kidn~y from donor
: animals sharing the same genetic makeup of the y~lk
sac donors. This raises the po~sibility of using
~HC-~ismatched yolk sac cells not only for
reconstitution purpos~s, but also as first step
T5TUTE ~ ET
WO93/02182 - 22 - PCT/US92/0~918
2 1. 3 .` ., .~
tolerogens for inducing specific tolerance in a
recipient for subsequent organ transplants.
In additi~n, this form of yolk sac cell
transplantation may be applied in situations where a
genetic defect has been det2cted in a fe~us. Human or
sther mammalian yolk sac cells carrying a normal wild
type gene or an exogenously introduced gene may be
injected into the developing f etus in a routine
pro~edure similar to that of ~mniocentesis ln utero.
lQ The gen~tic ~isorders for which this approach may be
: applicablP include, but are not limited ~o, sickle
cell anemia, thalassemia, and adenosine deaminase
~eficiency. Alternatively, yolk sac cells may be used
in ~ettings where a pregnant mother is dia~nosed to
carry ~IV, and reconstitution of ths fetus with yolk
a~ cells may prevent:viral infection of the fetus.
The ability of yolk sac cells to grow in
;~ xsnogeneic animals with no irradiation or chemical
treatment allows for large scale production o~ human
20 ~h~matopoietic ~ells and their secreted factors in
YL~- Human yolk sac cells may be injec~ed in a large
fa~rm animal, the blo~d collected, and large quantities
of~human proteins~or cells such as red blood cells,
: lymphocytes, granulocytes, platelets, monoclonal
antibodies and cy~okines purified for clinical use.
5.~5. BONE M~RROW REPLACEMENT THERAPY_IN HUMANS
: A protocol for the replacement of bone
marrow cell in human patients requiring bone marrow
transplantatlon may be devised using cultured human or
: ~ xenogeneic yolk sac cells. Yolk sac cells obtained
fr~m human yolk sac at day 10 of gestation may be
ola~ed using the procedures described herein,
expanded in culture, and cryogenically preserved as
donor cells for t~e transplant.
.
SII~T~TUTr SH~T
W~93/02182 2~ ~3~ J ~ pCT/US92/0591~
. Ablation of recipient patient bone marrow
cells may not be required, but if it is used, it can
be accomplished by standard total body irradiation
(Kim, et al., Radiology, 122:S23, 1977) or by
5 chemotherapy with a variety of commonly used compounds
including, but not limi~ed to Busulfan (Tutschka, et
al., 31Ood, 70:1382-1388, 1987), following the
conventional methods. Yolk sac cells can be
introduced into the recipient, using similar methods
0 for bone marrow cells. Prior to ln vivo transfer,
yolk sac cells may be transformed with a drug-
resistan~e gene, such as the methotrexate resistance
gene. This allows the subsequent use of high doses of
the corresponding chemotherapeutic drug to eradicate
~5 the less resistant host cells in a pa~ient, without
damage to the transferred yolk s~c cells. Post-
operative care would be the same as with
transplantation using bone marrow cells from a donor.
High doses of yolk sac cells obtained from
allogeneic or xenogeneic sources may be conti~uously
infused into a bone marrow transplant recipient in the
absence of prior chemotherapy or radiotherapy. This
presents a novel approach to bone marrow
transplantation without immunosuppressing the
recipient~
~ 5.6. IDENTIFICATION O~ NEW
: MARKERS ON YO~K SAC CELL5
Murine yolk sac cells express CD34 but none
of the other known leukocyte markers. It is possible
that yolk sac cells express other early markers which
have not yet been identified. If so, previous ~ailure
in identifying these unique molecules might be due to
their d~cr~ased expre~sion in more mature cells or
~ven stem cells after migration to other sit~ out of
the yolk sac. There*ore, yvlk sac cells may be used
~U~Ti,I~TE S~ET
WO93/0218. 2 ~ ~ 3; ~j 5 - 24 - PCT/US92/05918
to generate antibodies against their cell surface
antigens in order to identify and characterizs such
unknown markers.
Also within the scope of the invention is
the production of polyclonal and monoclonal antibodies
which recognize novel antigenic markers expressed by
yolk sac cells. Various procedures known in the art
may be u-~ed for the production of antibodies to yolk
sac cells;. For the production of antibodies, various
host animals can be i~munized by injection with viable
yolk sac cells, fixed cells or membrane preparations,
including1 but not limited to, tho~e of rabbits,
hamsters, mice, rats, etc. Various adjuvants may be
used to increase the immunological response, depending
on the host species, includin~ but not limited to
Freund's (complete and incomplete), mineral gels such
: as aluminum hydroxid&, surface aotive substances such
as lysolecithin, pluronic polyols, polyanions,
peptides, oil mulsions, keyhole limp.et hemocyanin,
dinitrophenol, and potentially useful human adjuvants
such a~ BCG (bacille Calmette Guerin) and
~ Corynebacterium ~arvum.
: Monoclonal antibodies to novel antigens on
yolk sac cells may be prepared by using any technique
which provid s for the production of antibody
molecules by continuous cell lines in cultur ~ These
include, but are not limited to, the hybridoma
techni~ue originally described by Kohler and Milstein
(1975, Nature 256, 495-497), the more recent human B-
cell hybr~doma technique (Kosbor et al., 1983,Immunology Today 4:72; Cote et al., l983; ProcO Natl.
Acad. Sci. ~0:2026-2030) and the EBV-hybridoma
technique ~Cole et al., 1985, Monoclonal Antibodies
and Canc~r Therapy, A~an R. Liss, Inc., pp. 77-96).
Techni~ues developed for the producti6n o~ ~'chimeric
TE S~E~
21 ' 3 ~
~93tO2182 - 25 - PCT/~'S92/05918
antibodies" by splicing the genes from a mouse
antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule can
be used (e.~., Morrison et al., 1984, Proc. NatlO
Acad. Sci., 81:6851-6~55; Neuberger et al~, 1984,
Nature, 312:604-608; Takeda et al., 1985, Nature
314 452-454)o In addition, technique~ described for
the production of single chain antibodies (U.S. Patent
4,946,778) can be adapted to produce single chain
10 antibodies.
Syngeneic, allogeneic, and xenogeneic hosts
may be immunized with yolk sac cells which can be
prepared in viable form, or in fixed form, or as
extracted membrane fragments. Monoclonal antibodies
can be screened differentially by selective binding to
yolk sac cells, but not to mature macrophages,
granulvcytes~ T, and B ells.
Antibody fragments which contain the binding
sit~ o~ the molecule may be generated by known
technigues~ For example, such fragments include but
are n~t limited to: ~he F(ab' )2 fragments which can be
produced by pepsin digestion of the a~tibody molecule
and the Fab ~ragments which can be generated by
: ~ reducing the disulfide bridge~ of the ~(ab') 2
: ~ 25 ~ragments.
6. EXAMPLE: GENERATION OF YOLK SAC
STEM CELLS FOR IN VIVO
HEMATOPOIETIC RECONSTITUTION
6.1. MATERIALS AND METHODS
6.1.1. ANIMA~S
BALB/c, C57BL/6 t beige nude X-linked
im~unodeficient (BNX), and C3H/SCID mice were
purchased from Jackson Laboratories (Bar Harbor, ME)
T~T~TE SaE~T
WO93/~2182 - 26 - PCT/US9~/05918
2 1 ~ 3 ~ ;J
and kept in the animal facility of Edison Animal
Biotechnology Center.
6.l.2. ISOLATION OF THE YOLK SAC
On d2y 7 of gestation (day of plug was
counted as day 0, female mice were sacrificed by
cervical dislocation, and uteri containing embryos
were placed in petri dishes with Dulbecco's Phosphate
Buffered ~aline (PBS) plus penicillin and streptomycin
antibiotics (~inal concentration :l000 units potassium
penicillin G and l000 ~g streptomycin sulfate/ml).
Under a laminar air-flow bench ,each uterine
segment containing an embryo was aseptically removed
by dissection with the aid of a dissenting microscope.
Each embryo surrounded by decidua capsularis was
transferred to another petri dish containing PBS plus
penicillin-streptomycin. The decidua capsularis was
opened with watchmakeris forceps and each embryo
transferred into an individual petri dish where yolk
sac tissue was excised from the amnion, placenta,
embryo, and Reichert's membrane in 0.02% EDT~ in PBS
at ~C for 15-30 minutes. The yolk sac cells in
single cell ~uspension were then washed in PBS before
culturing.
~5
6.l.3. CULTURE CONDITIONS
Disaggregated yolk sac cells were grswn in
alpha medium (Sigma) supplemented with l8~ :
heat-inactivated fetal calf serum, 0.2 ~m
~-mercaptoe~hanol, 50.~g/ml of gentamicin and 10% LIF
conditioned medium (medium of a LIF-producing cell
line, Cho LIFD at l00-lO00 u/ml). Cells were grown
without feeder layQrs on collagen or gelatin coaked
~ishe-~ and incubated at 37C in 5~ C2 in air. ~edia
were changed every other day.
SU~STITUT~ SHET
W~9~/02182 - 27 ~ 3 ~ PCT/US92/0591
6.l.4. FLOW CYTOMETRY ANALYSIS
l06 yolk sac cells were washed twice in cold
PBS containing O.l BSA and sodium azide. The cell
pellets were suspended in the same buffer containing
the test antibodies at 4C for 30 minutes. Cells were
then wash~d in cold PBS twice and analyzed by flow
cytometry. Antibodies specific for Thy-l, Ly-l, Ly 2,
Mac~ C class I and class II were purchased from
Boehringer Mannheim. Anti-Ml/70, anti-H2d and anti-H2b
antibodies w~re purchased from Pharmingen (San Diego,
CA~. Anti-CD34 was used as hybridoma supernatant.
Ç.l.5. NDUCTIO~ OF YOLK SAC DIFFERENTIATION
BALB/c yolk sac cells were grown to
l approximately 50% confluency in medium containing LIF.
The cell~ were harvested, washed and medium containing
growth fact~rs was a~ded. Growth factors used were:
LIF (l00-l000 U/ml), SCF (50 U/~13, EPO (1-25 U/ml);
IL-2 (l0-200 U/ml), an~ IL 3 (10-200 U/ml) in various
combinations. The medium wa~ changed every 2 days
until confluency was reached, at which time the yolk
: ~ sa~ cells were passed 1:4 into new gelatinized 35 mm
: culture dishes. At day 5, and 21, cells were prepared
for blood staining. Day 0, 5, and 2l cells were
~: 25 analyzed by flow cyto~etry for the appearance of
differentiated blood cells.
:: :
: 6.l.6. HEMAGGLUTINATION ASSAY
: Lipopoly~accharide (LPS) conjugated to
trinitrophenol (TNP) and human serum albumin (HSA)
c~njugated to TNP ~ere injected at 20 ~g/mouse
int~aperitoneally into BNX mice and SCID mice J
resp~ctively, both of which had previously received l06
~urine yolk sac cells intraperitoneally a month
earlier. A ~econd injection of the antigens waæ
T!T~T~ SH~ET
WO93/02182 - 28 - PCT/US92/0~g18
~113 i~,
performed one week later, animals were bled after
seven days and sera assayed for the presence of
specific antibodies.
A two~fold serial dilution of the mouse sera
was made in microtiter plates. Sheep red blood cells
(SRBC) coated with dinitrophenol (DNP) were added to
~ach well. The plates were incubated at room
temperat~re for one hour. The results of the assay
were a~sessed visually. A dif~used pattern of S~BC
indicated a positive TNP-specific antibody response.
Negative wells had a small, tight pellet o~ SRBC.
. 6.2. RESULTS
6O2.1. ISOLATION OF MURINE_YOLK SAC CELLS
In the:mouse, the yolk sac is fully f~rmed
by day 7 and blood island formation usually appears by
day 8.5 of gestation. Therefore, in order to isolate
~: ::; homogeneous and undifferentiated yolk sac cells, mouse
embryos were surgically removed prior to visible blood
island formation, preferably at day 7 of gestation.
: The yolk sac region of the embryos was separated by
excision, and the external surface of the yolk sac was
, .
immersed in cold EDTA~which caused the detachment of
25 ~:the yolk sac cells from the membrane into a single
cel~l suspension ~FIG.~ 1). When the physical
:; ~ appearance of yolk sac cells obtained from day 7 and
: day 8.5 embryos was compared by flow cytometry
analysis, freshly isolated day 7 cells clearly
displayed a much more uniform cell shape and cell size
than ~he day 8.5 cell:s, suggesting that yolk sac cells
~ were a homogeneou population at day 7 but by day 8.5,
: ~ifferentiative activities had already occurred to
generate a mixed population of cells in the yolk sac
(FIG. 2). There~ors, day 7 yolk sac cells were used
~ r ~EEl
W~93/02182 ~1~3 ~ Pcr/~ls9~/o59l~
for all in vitro and in vivo studies described herein,
infra~
6.2.2. CELL SURFACE PHENOTYPE
OF YOLK SAC CELLS
The freshly isolated yolk sac cells from day
7 mouse embryo-~ were immediately examined for their
cell surface expression of a number of known leukocyte
markers by reactivity with monoclonal antibodies.
~0 Such uncultured yolk sac cells express CD34 but not
Thy-l, MHC class I and class II antigens (FIG. 3).
Th~ expr~ssion of C~34 by yolk sac cell~ is consistent
with t~em being primitive stem cells as CD34 is
currently the earliest detectable marker on bone
marrow hematopoietic stem cells. The absence of MHC
antigen expression at this stage is significant in
that the likelih~od of rejection of these cells by a
: genetically di~parate host upon ln vivo transfer is
greatly reduced. ~urther, the lack o~ Thy-l
expression indicates that the yolk ~ac cells of the
invention represent an earlier cell population in
~ ontogeny than the Thy-l+ hematopoie~ic stem cells
: found~in bone marrow, thus should contain a
pluripotent population that is less committed to any
specific ~ell lineages.
6.2.3. LONG-TERM MAINTENANCE OF YOLK SAC CELLS
The yolk sac cells isolated from day 7
embryos were established in culture in the presence o~ -
3~ leukemia inhibitory factor (LIF) at l0-l00 U~ml
without a feeder layer. rhe cells expanded in number, .-
having a doubling time of about 18 hours. Such
cultured cells have been grown in vitro for over 4l
passages covexing a period of time over nine months in
35 c:on~inuous clllture. Alternatively"~olk sac cells
S~T~TUT~ SH~ET
WO93/021~2 30 _ PCT/US92/05918
2113'jC` i
could also be grown in stem cell factor with similar
results.
Although LIF is capable of suppressing
di~ferentiation of the yolk sac cells over an extended
period of in vitrQ yrowth, the effect of LIF is
incomplete because a small fraction of the cultured
cells beyan to express certain differentiation markers
including Thy-l and MHC-encoded molecules. However,
the majority o~ the long-term cultured cells retained
their original cell surfac~ phenotype. Further, such
cells con~inued to be pluripotent as evidenced by
their ability to give rise to mature blood cells in
vitro and in y~y~/ infra. The cells with the original
phenotype in long-term cultures may be obtained by
cel1 sorting or by repeated limiting dilution cloning.
: 6.2.4. DIF ERENTIATION OF YOLK SAC CEL~S IN VITRO
~ After one month of in vitro culture in the
:~ : presence o~ LIF, the yolk sac cells were tested for
their ability to differentiate into mature blood cells
of:all lineag~s in response to various known
hematopoietic growth factors including IL-3, IL-2, and
EPO.~ When cultured in IL-3 and EPO, the appearance of
: red blood cells:was readily detectable in the yolk sac
2~ cultures. In responce to CSF's and IL-3, the yolk sac
: cells matured into megakaryocytes and granulocytes.
:Fig. 4 is a blood stain of a yolk sac culture grown in
: ~ the:presence of:a~ombination of cytokines and the
: appearance of various blood cell lineages can be
identified, iIn addition, the expression of various
leukocyte surface markers by these cells became
detectable, including CD34, CD45, LFA, MAC-l, Ly-l and
Ly-2.
:
~ T~TUTE SHE~
3 ~
W~93/02182 - 31 - PCT/US92/05918
6.2.5. DIFFERENTIATION OF YOLK SAC CELLS IN VIVO
A long-term culture of yolk sac cells of
BALB/c origin was injected into allogeneic C3H/SCID
mice after 22 passages in vitro. Four weeks later,
spleens and livers of the treated animals were
analyzed for the presance of donor cells by monoclonal
antibodies.
The donor cells were identified by
antibodies specific for the donor H-2d haplotype.
Double staining eXperiments utilizing two antibodies
~ further demonstrated that certain subpopulations of
the donor cells expressed CD3, Thy-~, B220 and Ml/70
.(FXG. 5). Therefore, these results indicated that the
long-term cultured mouse yolk sac cells were capable
of di~ferentiating na~urally in vivo into T cells, B
cells and macrophages .
67 :2 . 6. GENERATION OF INMUNOCOMPETENT
CELLS BY YOLK SAC CELLS IN VIVO
2 0 In order to examine whe~he~ the yolk sac
cells could give rise to functionally mature blood
cells, the long term cultured yolk sac line was
transferred in vivo into allogeneic SCID or BNX mice,
: ~ and tested for spPcific antibody production. When the
25 ~B~X mice received yolk sac cells and subsequently were
: immunized with LPS a month later, specific antibody
tit~rs were detected in the sera (FIG. 6). As LPS is
a polyclonal B~cell activating agent, this result
shows the presence of functionally active
3~ antibody-producing B cells. Additionally, when SCID
mice were injected with yolk sac cells followed by HSA
im~unization, which is a T cell dependent antigen, an
antibody reeponse was again detectable, suggesting
that lony term cultured yolk sac cells could
diff~rentiate to become immuno-competent T and B cells
in vivo.
TITUTE S'~EET
WO93/02182 - 32 - PCT/US92/05918
9~
6.2.7. YOLK SAC CELLS REPOPULATE
CHEMO-ABLATED MOUSE SPLEENS
Certain classes of chemotherapeutic drugs
are effective, and have been used, as ablative agents
for bone-marrow in bone-marrow transplantation
procedures (Floersheim and Ruszkiewicz, 1969, Nature
222:854). One o~ the most effectiYe agents used t~
replace whole body irradiation in bone-marrow
transplantation procedures is the drug Busulfan
(~utschka et al., 1987, Blood 70:1382)~ Through
careful tItration of the dose of Busulfan and the use
of inbred lines of mice ~C57BL/6) of a defined age and
weigh~ (3-4 weeks of age~, doses o~ Busulfan have been
det~rmined which fully ablate the bone-marrow of these
mice but do not directly kill them. These doses of
Busulf an result in the sventual death of the treated
mice between 11 and 14 days if they do not receiYe
tran~p}anted bone-marrQw. This dose is 65 mg of
Busulfan/g:of body weigh~ administered in a single
dose by I.P. in3ection. When C57BL/6 mice were
treated:with this dose of Busulfan and then received
an I.Pv injection o~ 106 syngeneic cultured yolk-sac
cells 24 hrs. following Busulfan treatment, the
transplant recipients revealed spleen repopulation at
day 7 and:14 post Busulfan treatment (FIG. 7). On day
7,:spleen colony formation within the recipient was
observe~, indicative~of the initial stages of splenic
repopulation ~y~the~transplant. Additionally,
comparison at day 12 post treatment; of the spleens of
control ~usulfan treated mice not receiving yolk-sac
transplants and those~ animals receiving transplants
howed a~marked dif:ference in splenic viability.
~hile the spleens of control animals were dark, aimost
black in color, and appeared necrotic, the spleens of
transplant recipients displayed a red/pink color and
appeared normal and healthy. Further, the survival
SU~Ti ~ ~'TE S~E~r
~93/021X~ 2113 ~ ~ 5 PCT/US~2/0591~
time of the yolk sac cell-treated mice was extended to
between l8 and 20 days.
6.2.8. IN UTERO ADMINISTRATION OF
YOLK SAC CELLS RESULTS IN
TISSUE CH.IMERISM ____
A long-term cultured yolk sac cell line was
te~ted for its ability to survive in an allogeneic
ho~t~ l0,000-50,000 B~LB/c yolk sac cell~ after 13-20
passages in ~itro were injected in utero in day 8
embryos of C57BL/6 mics. At birth, the spleens and
livers of the neonates were harvested and analyzed for
the presence of donor cells.
Since the donor cells were of the H-2d
haplotype, a monoclonal antibody specific for H-2d
antigens was used to id~ntify the donor cells by flow
cytometry analysis. FIG. 8 presents the results from
two neonates examined and it clearly shows that donor
: cells were present in both he liver and spleen of the
: recipient mice in su~stantial number~. Therefore, in
terQ administration of yolk sac cells into
: MHC-mismatched mice resulted in tissue chimerism, and
: survi~al and homing of the cells to the
lymphohematopoietic organs. Tissue chimerism was
retained when the mouse tissues were examined even one
month after birth.
: 6.2.9. XENOGENEIC TRANSPLANTATION
: ~ OF YOLK SAC CELLS RE5ULTS
IN LONG-T~RM PERSISTENCE OF
~ELLS IN VIVO _ _
In order to test the feasibility of using
yolk sac ceIls in xenogeneic transplantation and
reconstitution, long-term cultured BALB/c yolk sac
cel1s were injected into a newborn Hampshire sheep and
a Nubian goat. The sheep receiv2d 40xl06 murine yolk
sac cells intravenously at day 3 after birth and the
S~TITUT~ SHEET
WO93/02182 _ 34 _ PCT/US92/0591~
2 1 ~ ~ 'J ~ j ;3
goat received the same cell dose at day 7 after birth.
Four days later, both animals received a second dose
of 200xl06 cells. After four additional days, a final
injection of 60xlO6 cells was given, the peripheral
5 blood mononuclear cells were harvested for antibody
staining and flow cytometry analysis about one and a
half month later.
FIG. 9 demonstrates that a substantial
n D ber of blood cells obtained from the sheep were
:~ lO reactive with anti-H-2d antibody. While there were
: ~ lower numbers of donors in the peripheral blood of the
goat, donor cells were nonetheless detectable. In
addition, cells expressing the murine T cell marker
Ly-l were also present from both animals. However,
15 neither animal had cells that were positive for the :;.
mu~ine macrophage marker Mac-l, consistent with the
:fact that macrophages are not normally present in the
peripheral blood.
The results~ of~this experiment are revealing
in a number of ways.; It illus~rates the possibility
of xenogene;ic reconstitution using murine yolk sac
cells:. Neither:animal was pre-treated with
i:rradiation or oytotoxic~drug. The high cell doses
and~the repetitive injections did not induce graft
25 ~rejection~. Both:~animals~also appeared normal and
heàlthy, having~;no~indication of graft versus host
reaction.~ The~consis~tent finding of a high number of
donor cells~recoverable~;from the sheep than the goat
: may be a result ~of~the goat being of an older age
before recei~ing:the first cell injection. The
yvunger age of the sheep when it was given the first
cell dose might have resulted in a more efficient.
~; induction of tolerance. However, there was still
: acceptance of the donor cells in the goat in the
: :35 absence of any prior immunosuppressive treatment. If
:
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2 1~ ~ 3 i~~
~'~ 93/0~182 PCT~'S92/0591
induction of tolerance is the mechanism underlying
this observation, this further sugyests that the
tolerized hosts may also accept other solid organs
including the heart, liver a~d kidney from xenogeneic
donors sharing the same haplotype of the original
donors. Finally, the expression of a T cell marker
indicates normal di~ferentiation and maturation in
yivo, and the absance of macrophages in the p~ripheral
blood suggests the appropriate homing of the right
cell lineages in the host upon intravenous
administration of yolk sac cells.
7. EXAMPLE: IN VIVO TRANSFORMATION
OF YOLK SAC_CELLS . _ :
In this example, both untransformed yolk sac
cells and a retroviral vector containing the exogenous
gene of interest are injected into the target animal.
The exogenous gene used is the growth hormone gene
~: (bGH~.
2~ Yolk sac cells harvested from day 8 C57~5JL
mouse embryos are cultured on an STO feeder layer
system until approximately 20 x l06 cells per culture
flask (l50 cm) were generated. Cells were passed to
new ~lasks when the cell density became greater than
80%. AlI experiments were performed with cells at
passage l0 or greater.
Newborn mice were injected I.P. with yolk
sac cells (2 4 x 106) at 3 to 5 days of age. Two
months [positive results have been obser~e~ with
infections as early as two weeks ~ollowing yolk sac
injection) after I.P. injection of yolk sac cells,
~ animals received lxl0~ viral particles of a replication
: deficient retroviral vector produced from the Moloney
murine L~kemia Virus based Mulligan ~2 packaging cell
line after tr~nsformation with the plasmid pLJPCKbGH
by ~.V. injection into the tail vein.
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W093/~2182 36 - PCT/~IS92/0591~
~113- ~rj
. Of 145 animals treat~d by this procedure,
112 were positive for bGH in the serum by ELISA assay.
The following is a breakdown of the positive bGH
levels of these test animals:
20-59 ng/ml = 56 mice
60-100 ng/ml = 20 mice
100-200 ng/ml - 14 mice
200-S00 ng/ml = 13 mice
> 500 ng/ml = 9 mice
.~
An equal number of control animals were
.injected with the retroviral vector but not with the
yolk sac cells. None of these were positive for bGH.
It is believ~d that either the retroviral
vector specifically can transfect the injected yolk
sa~ cells, and/or the yolk sac cells secrete f~ctors
having an effeot on the nearby cells, rendering them
susceptible to transfection.
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