Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ 93/18137 2 1 3 1 3 6 ~ PCT/US93/018~2
CU~T~RING OF ~ENATOPOIETIC 8TEM CELL8
~ND T~EIR GENETIC ENGINEER~NG
O~UC~ON
Technical ~ield
The field of this invention is expansion of human
hematopoietic stem cells and their genetic modification.
Baok~round
The availability of human hematopoietic stem cell
compositions offers a plethora of opportunities for
medicine. The hematopoietic stem cell is the progenitor
for all of the blood cells, including leukocytes,
including lymp~ocytic and myelomonocytic lineages, and
erythrocytes, as well as other t~pes of cells, such as
oste~clasts. There is the further possi~ility, that the
tem cell may also lead to stromal cells. These cells
1~ provide an eno~mous ~ange of functions. None of these
cells is believed to be capable of self-regeneration, 80
a~ to sur~iv~ during the li~etime of the host. The stem
cell i5 believed to be the only cell that is self-
r~g~nerating ~nd ~aint~ins $ts pluxipotent potential
during the life of th~ host. ~h¢refore, understanding the
role of the stem cell, the manner in which it regen~rateæ,
. . --
and the manner in which it i8 programmed to produce the
v~rious lineages will provide opportunitieæ for t~erapies
;;~ for a wi~e variety of`diseases.
2~ The a~ility to obtain substantially h~mogeneous human
~tem ~ell compositions offers new ~pproaches to bone
W093/18137 ~3~368 -2- PCT/~S93/018sZ~
marrow transplantation. Since there is evidence to
suggest that the stem cell will not be malignant, by
isolating human hematopoietic stem cells, one may avoid
restoring malignant cells to a host after radiation or
chemotherapeutic treatment of cancer or other malignancy.
The stem cells offer an entree to gene therapy, where the
modification will survive the life of the host. In
addition, by appropriate use of inducible promoters, one
can provide that expression of various protein products
may be achieved at ~elected levels of differentiation or
~ in selected lineagès, or even in response to particular
- ~ chemical clues, such as chemoattractants, particular
ligands, and the like. Also, as there is better
understandlng of the manner in which stem cells are
directed to specific lineages, there will be the
opportunity to produce in culture populations of
particular lineages, such as megakaryocyt~es, subsets ~f T
cells, monocyte , and the like.
An important aspect of this invention in the use of
stem cells will be the ability to expand the stem cells in
culture. Growing ~tem cells is different from growing
other cells, s~nce in order to expand ~t~m cellc, on- must
not only provide for r~generation, but inhibit the 1088 of
stem cell~ by diff~rentiation. The ~anner in which
2~ regeneration as against differentiation is regulated in
}~ bone marrow i~ not understood. Methods are therefore
,,
necessary which allow for long-term retention and
expansion of stem cell cultures.
~, .
~ 93/18137 2 1 3 1 3 6 8 PCT/US93~01852
Also, there has been no prior showing that one can
genetically engineer a stem cell. ~he unique
characteristics of the stem cell di~tinguish the stem cell
from other cell~ which have been ~uccessfully genetically
5 engineered. However, efforts to genetically engineer
progeny cells frequently encounter lack of transmission of
functional expression of the introduced construct in
progeny cells, lntermittent expression, and the like.
Therefore, even where one has demonstrated the successful
lo integration of the construct, there are instances
enoountered where subseguent growth of tbe progeny cells
and their differentiation resu~t in t~e failure of the
construct to function.
Relevant Literature
U.S. ~atent No. 5,061,620 describes a substantially
homogeneous human ~ematopoietic stem cell composition~and
the manner of obtaining such compo~ition. See also
r~f~rences clted therein. Stro~al cell-associated
~ematopoie~is is described by-Paul et al,, ~lood (199~),
77, 1723-1733. Murine leukemia ~nhibitory f~ctor is
taught to enhance retroviral-vector infection efficiency
of hematopoietic progenitors by Fletcher et ~1., Bl~od
(1990), 76, 1098-1103. Metcalf et ~1~, Blood, ~1990), 76,
2~ 50-56 de~cribes t~o f~ect of in~ected l-ukemia inhibitory
-~ f~ctor on bematopoietic and ot~er tissues in mice. The in
~itr~ e~fect of leukemia inhibitory factor on multi-
; potential human hematopoietic is described by Verfaillie
.~:
2 13 1~ 6 8 4 PCT/US93/0185~
; ~nd McGlave, Elood (1991), 77, 263-270. Dick et al.,Blood (1991), 78, 624-634 de~cribe gene transfer into
nor~al human bematopoietic cells employing in vitro ~nd in
~; vivo assays. The effect of leukemia inhibitory factor on
hematopoiesis is described by Metcalf, phil. Tr~ns. R.
Soc. Lond. (1990), B327, 99-109 and Leary et al ., Blood
(1990), ~5, 1960-1964.
A review of leukemia inhibitory factor (LIF) is found
in Metcalf, IntI. J. Cell Clon. (1991), 9, 95-108. A
de~cription of the effect of LIF on embryonic ~tem cells
is provided by Nichols et ~ y~ (1990), 110,
41-1348; Williams et al., ~u~e (1988), 336, 684-687
nd~Smith t ~ y~ (1988), 336, 688-690.
: ~ .
15 ~ SUMMARY OF THE INVENIION
th d d compo~itionC are provided for maintenance
nd~expansion of human hematopoietic ~tem cells in cul$ure
by using leukemia inhibitory factor (LIF) under conditions
~`~ where human hematopoietic 8tem cells are able to ~urvive
and to expand in culture. LIF may be used by itself or in
;~ co~bination with other added hem~topoietic cell factors in
n appsopriate culture ~edium. A substantially
~ooog~neou~ human hematopoietic ~tem cell composition i~
genetically modifiod u-ing appropriate DNA constructs for
2S introduct~on into t~ st ~ c-lls and ~nt gration. The
modlficat~ion of tb- ~t~ cells i6 d monstrated by
s-ays where long-term ~intenance of expression of the
~93/18137 2 1 3 1 3 6 8 PCT/US93/01~2
integrated gene is shown in a plurality of hematopoietic
lineages, both lymphocytic and myelomonocytic.
DESCRIPTION OF ~E ~E~I~1L~
Substantially homogeneous human hematopoietic stem
cells (hereinafter referred to as 'IhHSC'') are maintained
in long-term cultures and expanded in number in
appropriate media comprising added leukemia inhibitory
factor t"LIF"), optionally in conjunction with additional
added hematopoietic factors, under otherwise conventional
conditions. The hHSC may be maint~ined in culture for
long periods of time, as demonstrated by their capability
to continually differentiate into multilineage progeny.
The hHSC may be genetically modified by employing a
substantially homogeneoue stem cell composition with a DNA
construct providing a DNA ~equence of interest.
~articularly, a retroviral vector is employed for ,the
introduction~of the DNA construct; into the hHSC host. The
resulting cells may then be grown under conditions ~s
desari~ed for unmodified hHSC, where~y tha modified hHSC
~ay b2 expanded and used for a variety of purposes.
The hHSC which are employed ~ay be fresh, frozen, or
have ~een subject to prior culture. They may be fetal,
~eonate, adult, obtained from fetal liver, bone ~arrow,
2~ ~lood o~ ~ny other conventional source. The m~nner in
whic~ the stem cells ~re separated from other cells,
whether of the hematopoietic or of other lineage is not
critical to this invention~ Conveniently, the cells may
WO93/18137 PCT/US93/0185X
2 ~3 13 6 8 -6-
be separated as described in U.S. Pate~t No. 5,061,620.
As described, the substantially homogeneous composition of
hHSC may be obtained by selective isolation of cells free
of markers associated with differentiated cells, while
displaying epitopic characteristics associated with the
stem cells. The stem cells are characterized by both the
presence of markers ~ssociated with specific epitopic
~ites identified by antibodies and the absence of certain
markers as identified by the lack of binding of certain
antibodies. At such time as a specific marker is
identified for hHSC, binding of an antibody to such marker
may provide the desired composition.
A large proportion of the differentiated cells may be
removed by initially using a relatively crude separation,
l~ where ma~or cell population lineages of the hematopoietic
system, such as lymphocytic and myelomonocytic, are
removed, as well as minor populations, such' as
megakaryocytic, mast cells, eosinophils and baæophils.
Usually, at least about 70 to 90 p~raent of the
~ematopoietic cells will be removed. If desired, a prior
~eparation may be employed to remove erythrocytes, by
e~ploying ficoll-hypaque ~eparation.
The gross separ~tion may be ~chieved using ma~netic
beads, cytotoxic agents, affinity chrom~tography, panning,
2~ or the like. Antibodies which find use include ~ntibodies
~ of CD34, Class II HLA or other marXer which allows for
:~ removal of most, if not all, mature cells, while being
~ a~sent on hHSC.
2131368 PC~IU~93/ ~1 852
IP~WS 2 2 FEB l994
Concomitantly or subsequent to the gross separation,
which provides for positive selection, a negative
selection may be carried out, where antibodies to
specific markers present on dedicated cells are employed.
S For the most part, these markers will include CD3, CD7,
CD8, CD10, CD14, CD15, CD19, CD20, CD33, preferably
including at least CD3, CD8, CD10, C~19, CD20, CD~3,
normally including at least CD10, CD19, CD33. The
hematopoietic cell composition substantially depleted of
dedicated cells may then be further separated using a
marker for Thy-1, whereby a subst~ntially homogeneous
stem cell population is achieved. Exemplary of this stem
cell population is a population which is CD34+ Thy-1+,
which approximates the substantially homogeneous stem
cell composition.
The hHSC composition is characterized by being able
to be maintained in culture for extended periods of time,
being capable of selection and txansfer to secondary a~d
higher order cultures, and being capable of
differentiating into the various lymphocytic and
myelomonocytic lineages, particularly 8- and
T-lymphocytes, monocytes, macrophages, neu~rophils,
erythrocytes, and the like.
A pluripotent human stem cells may be defined as
2s follows: (1) gives rise to progeny all defined hemato-
lymphoid lineages; and (2~ limiting numbers of cells are
capable of fully reconstituting an seriously
.~
immunocompromised human host in all blood cell types and
AME~O~o sHEE~
WO93/18137 - PCT/US93/018~
~, ~3~36~ -8-
their pro~enitors, including the pluripotent hematopoietic
stem cell by cell renewal.
The hHSC are then grown in culture in an appropriate
nutrient medium, which medium may be a conditioned medium,
S a co-culture with an appropriate stromal cell line, or a
medium comprising a ~ynthetic combination of growth
factors which are sufficient to maintain the growth of
hematopoietic c-lls.
For conditioned media or co-culturest various stromal
cell lines may be used, since it is found that human
stromal cell lines are not required. Thus, other stromal
cell lines may be employed, such as rodentiae,
:Y ~
; particularly murine~ A number of murine stromal cell
lines are desoribed in Whitlock et ~1., Cell (1987), 48,
1009-1021, AC6.21 being deposited at the ATCC as
. Other: stromal cell lines may be
developed, if desired.
Various deivices exist for the co-culture which allow
for growth and maintenance of cells. Thus, devices
employing crossed threads, membranas, controlled medium
flow, and the like may be employed for the growth of ~he
cells for removal of wa~te products, and repleni~bment of
the various factors as~ociated with cell growth.
Conven~ently, tis~u- cul~ur- pl~tes or flasks m~y bei
2~ employed where confluent stromal cell layers may be
~aintained for extended periods of time without passage,
, - ~
but with changinq of the tissue culture medium about every
fi~e to seven days.
;: ~
~`JO93/~8137 2 1 31 3 6 ~ PCT/US93/018~2
The hHSC may be grown in co-culture by placing the
hHSC onto the stromal cell lines, either directly or
separated ~y a porous membrane. For example, ~or- about
3x104 to 3x105 cellslml are placed on a confluent stromal
S cell layer. The media employed in the coculture may be
any convenient growth medium, such as RPMI-1640, IMDM,
etc. either individually or in combination, where
~ppropriate antibiotics to prevent bacterial growth and
other additives, such as pyruvate (O.l-5 mM), glutamine
lO(0.5-5 m~), 2-mercaptoethanol (l-lOx105 ~) and from about
5-15%, preferably about 10% of serum, e.g. fetal calf
serum.
In addition to the other additives, LIF is added in
from bout l ng/ml to lO0 ng/ml, more usu~lly 5 ng/ml to
30 ng/ml. Other factors may also be ineluded, such as
inte~leukins, colony stimulating ~actors, steel factor, or
the like. Of particular interest in addition to LIF are
IL-3, IL-6, ~nd G~-CSF.
The factors whirh are employed may be naturally
occurring or synthatic ~g. prep~red r~c~mbinantly, and
~ay ~e human ~r of other 8pe~ies, ~gO murine, pra~erably
, human.
- The amount o~ the other factors will generally be in
the range of about 1 ng/ml to lO0 ng/ml. Generally, for
IL-3, the concentration w~ll be in the range of about
~ ng~ml to S0 ngtml, more usually 5 ng/ml to lO0 ng/ml;
: fQr IL-6, the concentration will be in the range of about
5 ng/ml to ~0 ng/ml, more usually 5 ng/ml to 20 ~g/ml, and
WO93/18137 PCT/US93/0185~
~3~3~ o-
for GM-CSF, the c~ncentration will generally be 5 ng/ml to
50 ng/ml, more usually 5 ngtml to 20 ng/ml.
The LIF and other factors may be present only during
the initial course of the stem cell growth and expansion,
usually at least 24 hoùrs, more usually at least about 48
hours or may be maintained during the course of the
expansion. Thus, it is found that significant effect can
be achieved by exposure of the hHSC initially in the
growth medium, without maintaining the concentration
during the entire course of the expansion.
For genetic modification, the cells may be grown for
sufficient time to reacb the desired population level.
;~ Usually, ~t lea~t ~Xl0~ hHSC cells will be present,
preferably lx105 cells. LIF will be present at lQaSt
1~ initially in the culture medium, usually for at least 12,
more usually at least 24 h, where exposure to LIF ~nd
otber growth factor~ may be substantially terminated. ~IF
~nd optionally the other growth Xactors may be ~aintained
during the course of the grow~h of the cells. The cells
will have been grown for at le~st l2, usually 24 hours, in
the medium with the factors, before contact with the DNA
construct. For genetic modification of the hHSC, usually
a retroviral vector will be employed.
Various retroviral ~-ctors may be employed for
2~ genetic modifioation. One will normally use combinations
of retroviruses and an appropriate pack~ging line, where
the capsid proteins will be functional for infecting human
; cells. Various amphotropic virus-producing cell lines are
.
- ' , 2l31368
~O 93/18137 pcr/us93/o1852
--11--
known, such as PA12 (Miller et al., Mol. Cell._ Biol. 5
(1985), 431-437), PA317 (Miller et al., MQ1 Cell. Biol.
Ç (1986), 2895-2902) GRIP (Danos et al., PNAS 85 (1988),
6460-6464). Usually, the cells and virus will be
incubated for at least about 24 hours in the culture
medium. The cells are then allowed to grow in the culture
~edium for at least two weeks, and may be allowed to grow
for five weeks or ~ore, before analy is.
The constructs which will be employed will normally
, ~
10 include a marker, which allows for selection of cells into
which the DNA has~been integrated, as against cells which
have not integrated the DNA construct. Various markers
exi~t, particularly antibiotic resistance marXers, such as
resistance to G418, hygromycin, and the like. Less
15 conveniently, negative selection may be used, where the
marker is the HSy-tk gene, which will make the cells
ensitive to agents, cuch as acyclovir and gancyclovi~.
The l:onstructs can be prepared in a variety of
~:onventional ways. Numerous vectors are now available
20 which provide for the desired features, such aæ long
terminal ~epeats, marke~ genes, and r~strict~on ~ites.
- Thu~, one ~aay ~ntroduce tbe vector in an appropxiate
plasmid and manipulate the vector by re~triction,
in~ertion of the d-sired gene, with appropri~te
~; 25 tr~cription~l and translational initiation and
~y ~ terl-ination r-qions, and then introduce the plasmid into
- ~ an ~ppropriate packaging host. Thus, at each of the
. .
D~nipulations, one may grow the plasmid in an appropriate
~3~ PCT/US93/0185~2
-12-
prokaryotic host, analyze th~ construct to ensure that the
desired construct has been obtained, and then subject the
construct to further manipulation. When completed, the
plasmid or excised virus may then be introduced into the
packaging host for packaging and isolation of virus
particles for use in the genetic modification.
The introduction of DNA can be used for a wide
variety of purposes, such as gene tharapy, introduction of
novel capabilities into the hHSC, direct dedication to a
particular lineage or subset of such lineage, enhancement
of maturation to a particular lineage or subset, or the
like. In view of the ma~or role hematopoietic cells play
in the functioning of the human host, their wide-spread
presence, and their varied capabilities, the hHSC have
great therapeutic potential.
There are many genetic diseases specific for
hematopoietic cells, including ~ickle cell ane~ia,
~ thalassemia, thrombocytopenia, hemophilia, combined
i~munodeficiency, and most leukemias. For the most part,
these diseases may be treated by homologous recombination,
where at least one copy of the defective gene may be
modified to the wild-type or a ~unctioning gene. For the
mo~t part, it will not be nec~s~ary to correct both
copies, usually correcting one copy sufficing to provide
for therapeutic trQatment. There are numerous
descriptions of methods of homologous recombination in the
literature, see for example, Mansour, et al., ~ure
`~'093/18137 2 1 3 1 3 6 8 PCT/US93/018~2
-13-
(1988) 336, 348-352 and Schwartzberg, et al., ~NAS USA
(199o) 87, 3210-3214.
Alternatively, one may use the hHsc and their progeny
as carriers for the production of a wide variety o~
products, where the host is genetically deficient or as a
result of a ~ubsequent disease has become genetically
deficient. Genetic diseases involving lack of a
particular natural product include muscular dystrophy,
where there is a lack of dystrophin, cystic fibrosis,
Alzheimer's disease, Gaucher disease, etc. In those
instances where a particular polymorphi~ region of a
polymorphic protein such as a T-cell receptor, major
hi~tocompatibility complex antigen, or immunoglobulin
~u~unit is involved with susceptibility to a particular
disea~e, e.g. an autoimmune disea~e, the particular exon
may be "knocked out" by homologous re~ombination, so as to
provide hematopoietic ~ells which will not be respon~ive
to the di~ease.
In other situations, such as diabetes, where cells
have ~een de~troyed as a result of autoimmunity, the hHSC
may be modified to provide for cell& which will respond to
~he ~eed $or ~ecretion o~ insulin, where appropriate
enhancers and promoter~ may be employed, so as to have the
insulin production regulated in analogous manner to the
r gulatlon in th~ ts of Langerhan~. ~hl- may involve
the expression of the insulin receptor in an appropriate
~ematopoietic lineage.
wog3/2 ~ ~3 6~ PCT/US93/018
-14-
Also, multiple drug resistance gene(s), e.g. Da~
may be introduced to protect the cells against cytotoxic
drugs, transcription of ribozymes to protect against viral
infection, or expression of ~arious protein products to
inhibit viral replication intracellularly, e.g. the tat
gene with ~IV.
` Modified ~tem cells may also be found for use in the
treatment of aging, autoimmune diseases, hematopoietic
disorders, and viral infections.
In many situations the therapy involves removal of
bone marrow or other source of stem cells from a human
host, isolating the stem cells from the source and
expanding the ~tem cells. Meanwhile, the host may be
treated to su~stantially or complete ablate native
hematopoietic capability. The stem cells may be modified
during this period of time, so as to provide for stem
cells ha~ing the desired genetic modification. A~ter
completion of the treatment of the host, the modified stem
cells ~ay then be r~stored to the host to provide for the
new capability. If necessary, the process may be repe~ted
to ensure the ~ubætantial absence of the original`~tem
~ells and the substantial population of th~ modified ~tem
cell~.
To prove that one h~s the modi~ied stem cells,
~ 2~ ~arious techniques may be employed. The genome of the
- - cells may be restricted and used with or without
mplification. The polymerase chain reaction, gel
electrophoresis, restriction analysis, Souther~, Northern,
~"~93/18137 2 1 3 1 3 6 8 PCT/USg3/018S2
and Western blots may be employed, sequencing, or the
like, may all be employed with advantage. In addition,
the cells may be grown under various conditions to ensure
that the cells are capable of maturation to all of the
hematopoietic lineages while maintaining the capability,
as appropriate, of the introduced DNA. Various tests in
vitro and in vivo may be employed to ensure that the
pluripotent capability of the stem cells has been
maintained.
To demonstrate differentiation to T cells, one may
isolate fetal thymus and culture the thymus for from 4-7
days at about 25C, ~o as to substantially deplete the
lymphoid population of the fetal thymus. The cells to be
~.,, ~,
te~ted are then microinjected into the thymus tissue,
~ lS where the ~LA of the population which i8 injected is
- ~ ~ismatched with the HLA of the thymus cells. The thymus
tissue may then be transplanted into a ~515L~Eil mouse~as
deseribed in EPA 0 322 240, where ~he thymus is
conveniently transplanted ~nto the kidney capsule.
For erythrocytes, one may use oonventional techniques
to identify BFU-E units for example ~ethylcellulose
~ulture (Metcalf, In: Reçent Results in C~ncer Res.
~1377), 61. Spinger-Verlag, 8erlin, pp. 1-22~)
demonstsating that the cells are capable of developing the
~ 25 rythrO~a lineage.
~ n identifying myeloid and B cell capability,
~.
conveniently, the population to be tested is introduced
fir~t into a hydrocortisone containing culture and allowed
W093/1;137~ PCT/US93/018~
to grow for six weeks in such culture. The medium
employed will comprise a 50:50 ~ixture of RPMIl640 and
IMDM containing 10% FCS, 10% horse serum, 50 ~g/ml
streptomycin/penicillin, glutamine and 5xlO~ M
hydroc~rtisone. During the six-week period, it will be
anticipated that in the absence of progenitor cells, all
of the mature cells would die. If at the end of six
weeks, myeloid cells are still observed, one m~y conclude
that there is a progenitor cell whi~h is providing for the
continuous differentiation to myeloid cells.
~t thi~ time, one may then change the medium, so that
~ ~ the medium now lacks hydrocortisone, to encourage the
.~ . . .
- ~ growth of B cells. By waiting 3-~ weekæ and demonstratinq
the presence of B cells by FACS analysis or other
analytical procedure, one may conclude that the progenitor
. . ~
cells which previously were capable of producing myeloid
~ ~ cells axe also capable of producing B cells.
; Human hematopoietic cells grown in the presence of
hydrocortisone can be maintained for at least four months.
Similarly, human hematopoietic cell cultures can be grown
in the absence of hydrocortiæone for at least four months,
which cultures will contain B lymphocyte~ as well as
~yelomonocytic cells. One may then ~ort the cell cultures
for identi~cation of hHSC.
The ~t~m cells may be administ-red in any
physiologically acceptable ~edium, normally
intravascularly, ~lthough they may also be introduced into
one or other convenient site, where the cellæ may find an
::
2131368
'~'093/18137 PCT/US93/01852
-17-
appropriate site for regeneration and differentiation.
Usually, at least lx105 cells will be administered,
preferably lxlO6 or more. The cells may be introduced by
injection, catheter, or the like. If desired, depending
S upon the purpose of the introduction of the cells, factors
may also be included, such as the interleukins, e.g. IL-2,
IL-3, IL-6, and IL-ll, as well as the other interleukins,
the colony stimu~ating factors, ~uch as G-, M- and GM-CSF,
~nterferons, e.g. ~-interferon, erythropoietin, etc. The
amount of these ~arious factors will depend upon the
purpose of the administration of the cells, the particular
~ needs of the patient, and will normally be determined
;~ empirically.
~ The stem cell compositions which are employed will
;; l5 generally ha~e fewer than 5~ of lineage committed cells
and wi~l ~e capable of cell-free generation in a co-
culture m~dium and differentiatio~ to member~ of ~the
.~ .
ly~phoid and myelomonocytic hematopoietic lineages. They
will generally ~ave at least 80~ of the cells
c~aracterized ~y being human, bematopoietic, and being
CD34~ lO- l9- 33 and Thy-l~. In addition, they may when
stained with rhodamine, be either rhodamine ~igh or
rhodamine low or a combination thereof. Preferably, ~he
cell~ will be shodamine low. See Sp~ngrude, ~ ol.
Todav 1~989), 344-350. The cells may be frozan at l~guid
nitrogen temperatures ~nd stored for long periods of time,
~eing thawed and capable of being reused. The cells will
usually ~e stored in ~ 10% DMS0, 50% FCS, 40% RPMIl640
:
~3~3 -18- PCT/US93/0l8~
medium. Once thawed, the cells may be expanded by use of
growth factors and/or ctromal cells associated with stem
cell proliferation and differentiation.
The following examples are offered by way of
illustration and not by way of limitation.
EXPERIME~TAL
Materials and Methods
Antibodies. The antibodies to CD34 were obtained
from I.D. Bernstein (Andrews et al., Blo~d (1986), 68,
1030); t~e antibody for Thy-l was obtained from Fabre
~Dalchau and Fabre, ~. Ex~. Med. ~l979), 149, 576). The
antibodies for CD34 were detected using the appropriate
anti-Ig conjugated to fluorescein, phycoerythin, or Texas
red ~Cal Tag) or bound to magnetic beads (Applied Immune
Sciencès tAIS]) and separated magnetically. The Thy-l
antibody was a fluorescein, phycoerythrin or biotin
conjugate, where the biotin conjugate was detected with
Texa~ red-avidin (Cal Tag).
'5t~ c~l~;. A Becton-Dickinson F~CS ~odified as
descr~ed (Parks and Herzenberg, M~enh ~ : eD:Ll (1984),
108, 197) was e~ployed. The du~l la~er in~trument allows
for four fluorescent par~meters and two lig~t scatter
2~ p~r~eters to be recorded for each analyzed cell.
; Residual erythrocytes and dead cell and debris were
excluded from analysis by light scattering gating and
propidium iodide (PI) staining or by scattering alone in
2131368
~'~93/18137 - PCT/US93/01852
- --19--
four color analyses. Compensation for spatial overlaps of
fluorescein and phycoerytherin and fluorescein and
propidium iodide was adjusted electronically.
For cell sorting, the stained ~amples were maintaine~
at 40C throughout the sorting procedure. Sorted drops
were collected in RPMIl640 containing lO~ fetal calf
~erum. Following isolation of a cell population by F~CS,
the sample was diluted l:l in HBSS, centrifuged for lO min
at a rcf of 200 and resuspended in 50 or lO0 ~l of HBSS
for hemocytometer counting.
The culture assays were performed as follows: AC6.21
confluent stromal cell layers were maintained for up to
3-4 weeks without passage by .changing of the tissue
culture medium every 5-7 days. To passage, the stomal
lS cell layers were washed three times with serum-free
medium, then overlaid with 2.5 ml (T~25 flask) of
0.5 mg/ml collagenasedispase (Boehringer-Mannheim,
Indianapolis, IN) in ~erum-free medium. The cultures were
~llowed to incubate 15-30 min at 37C; then the cells in
the enzyme-containing ~edium were collected in RPMIl640
~edium with serum ~dded. The stro~al cells were ~uspended
by pipetting with ~ ~steur pipet, then cultured directly
~t one-fifth to one-fiftieth of the original cell
concentration. Generally, confluent ~tromal layers
2~ ~ubcultured at l:lO r~ached confluency again at after 5-7
day~. Subclones were obtained by limiting dilution
ulture from 30 to 0.3 cells per well.
~:
.~ ~
~3~3 zo PCT/US93/01852
Cell suspensions of human fetal bone marrow were
prepared from long bones of fetuses from 16-20 week
gestation. The bones are split lengthwise and the
medullary cavity is ~craped with a scalpel blade. The
S bones are then placed in a l mg/ml solution of
collagenase-dispase in RPMIl640. The bones are incubated
for 30 min at 37~C, after which time the medullary cavity
is flushed with media ~RPMIl640 with pen/strep, 2-ME and
5% FCS) to remove hematopoietic cells. Alternatively,
~one marrow may be flushed from the marrow cavity without
the collagenase-dispase treatment.
Cell suspensions are prepared from livers of 16-20
week gestation fetuses. The liver is minced and then
pipetted to release cells. The cell suspension is then
placed on Ficoll gradient to remove hepatocytes, red blood
cells and debris. The hematopoietic dells are then
harvested.
Adult bone marrow i8 obtained from aspirates, which
a~e tre~ted to remove red blood aell~ ~efore use.
~ulk cultures are obtained by placing t~e human cells
.,
on t~ previously e~tabli~hed confluent l~yer of ~ouse
stromal cell lines~ From 3x104 to 2x105 cells per ml are
placed on the stromal cells in either ~-25 flasks or six-
well plates, by ~ddition of 3 ml to e~ch well of a ~ix-
well plate or 5 ml to ~ T-25 flask. A 50:50 mixture of
RPNIl64n and IMDM containing 50 U/ml penicillin/50 ~g/ml
streptomycin, 1 mM sodium pyruvate, ~ mM glutamine, 5X105 M
2-mercaptoethanol and ~0% fetal calf serum is employed.
wog3/18137 2131 3 6 8 PCT/US93/01852
-21-
For Dexter-type conditions, IMDM containing 50 V/ml
penicillin/50 ~g/ml strept~mycin, 1 mM sodium pyruvate,
2 mM glutamine, 10% fetal calf serum, 20~ horse serum and
lQ~ N hydrocortisone sodium suc~inate is employed. 80ne
marrow cells grown in the Dexter-type medium give rise
only to myeloid differentiation. Cultures were
established with whole-cell populations or cells
fractionated ~y their expression of cell surface antigens
(CD34, Thy-l).
~ne can determine the frequency of cells in the
startinq population which grow under the above-defined
conditions. The frequenc~ is determined by the cell
number ~t which 37% of the wells show no growth.
The E~fect Qf Growth Factors Qn Cell G~Qw~h. Into
1~ 10 ml of long-term culture medium was added lOO ~l (about
~ ,:
lOO cells) per well in a 96 well plate, where each of the
wells had a ~on~luent ~tromal monolayer of AC6.2l. On~-
~alf of the medium was replaced with fresh long-t~rm
culture ~ediu~ every week. Different factors were added,
i~di~idually or in combination, where the amount added was
the opti~um concentration to maximize the number of cells
~btained under the above conditions in 35 days. The
factors employed were IL-3, I~-6, GM-CSF, steel factor
(S~) and ~I~. Thes- f~otor~ were obtained as lyophilized
2~ recom~inant proteins (powdes) from R~D Systems
(Minneapolis). At the end of various time periods, lO-l5
wells were screened ~y FACS analysis and the total number
WO93/18137 PCT/US93/018~
~3~3~ -2~-
of cells counted. The following table (Table I) indicates
the results.
wo 93/18137 2 1 3 1 3 li 8 PCI/US93/01852
23 o
Ioooooo
Q ~ O O O O .
I O O O O O O 3
I ~ ~ U') U~
¦ A A A A A O O
. 3
I ~
U o o o ~
I ~ O O U
E ~ ~~ .C
'E~
~ o o 3
: ~ ~ ~ ~
I o o
.~.
U
O
~o
+ +
~D ~D ~ O
U ~ + + ~ ~ ~
I + ~q ~ ~ ~
~ ~ ~ + ~ + + ~ ~ @
I O E ~ H 1~ 1 ~ O ~
,. E~ 5~ 1 z ~
WO93/18137 PCT/US93/0185~
~3~36~ - 24 - `
In the next study, the presence of cells having
various markers was determined. CD7 is a T-cell marker
and not unexpectedly, T-cells were not observed, since
there is no exposure to a thymic environment. CDl5
indicates monocytes and granulocytes, CDl9 indicates
B-cells and CD33 indicates myeloid cells. By frequency is
intended the number of wells in which cells carrying the
markers were observed of 15 wells (Table II).
`:~
~ ~ .
.
, ,~, ~ .
,, i :
,,
~'`'O 93/18137 2 ~ 131 3 6 8 P~/US93/01852
____
U
~ ~ ~ .
I ,~
t~
WOs3/18137 PCT/US9310185~
~3~36~ - 26 -
In the next study, a FACS analysis was made of the
CD34, Thy-l populations after 5 weeks in the above
culture. The next table shows the percentage of CD34
Thy-l+ populations after S weeks and the num~er of wells
which were positive of the 15 wells analyzed by FACS
(Table III).
wo 93/18~37 2 1 3 1 3 6 8 PCI/US93/01852
CC
~ :: ~1
:c ~ ~n ~:
O ~ ~
., P ~ ~ O ~ 1 ~ a
C:~ ~ ~
V ~ I~ ~ I ~
s3 I ~ ~ ~ ~ 8`
H ~ ~ C ~ 1~
~ E ~ C
oq 1~: ~ o It~
~ ~ 3 ,~ C u~
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U~ C.~ ~
N
e
~n
~: X
+ + +
~ ~ +
f ~ + ~ +
~ ~ ~ ~ &.
E~
.
u~ o In
., .
W O 93/18137 P(~r/US93/018~2
~3~36~ - 28 -
-
In the next study, the above study was repeated,
except that the culture was maintained for seven weeks.
The f ollowing table indicates the results (Table IV) . ~
) 93/18~37 2 1 3 1 3 6 8 PCI`/US93/01852
~ ~1
~ ~- ~
~ ~ .
~ ~ O V
+ ~ i~
C~ ~ p . ~
`:~
U~ o
WO93/18137 - PCT/US93/01852
~3~3~ 30 -
From the above results the following conclusions are
that the stem cell populations in the in vitro cultures
have the right surface markers and the right cell size and
granularity (Paint-a-Gate).
To further demonstrate the activity of the CD34 Thy-l
population after in vitro culture, secondary in vitro
cultures were prepared. The cells from 50 wells of the 24
hour treatment wi~h LIF, IL-3 and IL-6 after 5 weeks were
sorted for the CD34 Thy-l populations. The total cells
were about 5xl06. After sorting, about 5x105 of CD34 Thy-l
cells were obtained. These were promptly introduced into
wells comprising confluent cultures of AC6.2l with long-
term culture medium at about lO0 cells per well. The
following table indicates the results.
213136~
~"1) 93/18137 3 1 PCI`/U~i93/01852
~ :~ .~ I ~ ) O ~
C~ U ~ ~ ~ :~
1~ ~ ~ ~ h ~ ~ h
U
C~ ~ I ~ -I
l ~ N
~:1 .
+
E t- I
U~ o U~
~ ~ .
W0~3/18137 3 2 P~/US93tOl~
'1.~3~' 6
~ C~ .~ ~ ~ ~
I o
~ 3
Z ~ Ul U~ U~
;~ C . ~o ~ ~
+
+ + +
o ~ ~ ~
H H H
.
"'~93/18137 2 1 ~ 1 3 6 8 PCT/US93/01852
- 33 -
Infection of CP34~ Thy-l+_po~ulation. The amphotropic
retroviral vector ~Mo+PyFlOl-~ was employed. Valerio et
al., Gene (1989), 84, 419-427; and Bambesechem et al., J.
EXD. Med. (l990), 172, 729-736. The infection was
performed by introducing approximately lO~ CD34 Thy-l~
cells and lxl06 CFU of the virus in l ml of long-term
culture medium ("LTCM") comprising lO ng/ml each of LIF,
IL-3, IL-6, ~nd GM-CSF. The cell mixture was maintained
for 24 hoùrs, and the medium diluted to lO ml with LTCM
and lO0 ~l of the medium introduced into wells in which
confluent layers of the AC6.21 stromal line was present.
The cells from 5-l5 wells, each of the cells with and
without virus, were then introduced into a methylcellulose
culture and maintained for 2 weeks. At the end of this
l~ time, all cells were collected from each methylcellulose
: culture and analyzed by DNA PCR for the ~eo gene.
In a second series of experiments, to the methy~-
cellulose culture was added G418 at l ~g/ml and cells in
~ wells, which had not been contacted with the virus, and
cells in 20 wells which had been contacted with ~he virus,
were prepared. The cultures were maintained for 2 weeks,
after w~ich all of the cell~ from e~ch cu~ture wexe
isolated and subject t~ the ~ame ~nalysis ~or the n~Q
gene. Also, the cells were analyzed for the presence of
2~ the polyoma enhancer sequence.
W093/18137 PCT/US93/0185~
~3~36~ - 34 -
The primers employed are as follows:
CA TCGCATGAG CGAGCACGTA (SEQ. ID NO:l) Neo-l
CGATGCCTGC TTGCCGAATA TCATG (SEQ. ID N0:2) Neo-2
CTAGACTGG CCGTGCGACA TCCTCT (SEQ. ID NO:3) PyFlOl-3
CAAT CATTACTATG ACAACAGTCT AG (SEQ. ID N0:4) PyFlOl-4
T~e anticipated fragment with primers for the neo
gene was 180 bp. Samples from cells which had not been
selected with G418, and which were not exposed to the
virus showed no band, while 3 samples from cells which had
been exposed to the virus were positive. The negative and
positive controls for PCR were as expected. A 2% agarose
gel was employed for electrophoretic separation.
In the electrophoresis for cells which had been
exposed to the virus, the 3 control samples of cells which
~ad not been exposed to the virus were all negative, while
cells from 12 wells, where the cells had been exposed to
the virus were all positive.
To further establish the absence of any artifacts,
the PCR produc~s were digested with restriction enzyme
SphI which provides 120 bp and 60 bp fragments, or NcoI
which provides l50 and 30 bp fragments. The resulting
gels provided the anticipated bands; where aells which had
not ~een infected with virus were negative, while cells
which had been infected with virus showed 2 smaller bands
than the original band.
In the next ~tudy, t~e unin$ected cells and infected
cell~ were studied to determine their ability to provide
long-term T-cell reconstitution in the thymus of the SCID-
~ , . . .. . .
2l3l36~
`~'~93/18137 PCT/US93/01852- 35 -
hu/thymus model, where fetal thymus is introduced into thekidney capsule of a C.B.17 scid/scid mouse. The cells
which are employed for culture differ in HLA from the
cells of the thymus. (See PCT/US9l/02373 for a
description of the test procedure.) lO~ CD34 Thy-l cells
from five-week cultures, where the cells had originally
been exposed for 24 hours to LIF plus IL-3 plus IL-6 or
these factors plus virus as described previously and
;~ allowed to grow for five weeks, were employed. At the end
of this time, the cells were sorted for CD34 Thy-l for
injection into the thymus. FACS analysis showed the
pre~ence of T-cells ob erved by markers for the surface
mbran~ proteins CD3, CD4 and CD8 in conjunction with a
~ar~er for the HLA of the donor cells in two or three
tudie~. Similarly, when the same T-cell markers were
employed as against an antibody for all human cells, in
the ame two or three experiments, a large population of
T-cells were observed.
The cells in the thymus were isolated and subjected
to DNA ~CR analysis. Where the pri.mers employed were for
the ~eo gene, employing a total of 106 cells from the
thymus, a control mouse gave a negative result, while a
po~itive result is observed under the following
conditions: 3 months ~fter injection CD34~ Thy-l~ positive
2~ cells from a secondary culture at lO~ cells/thymus; 2.5
~- ~ont~s after in~ection of CD34~ Thy-l~ positive cells from
a prim~ry~ culture, 2x103 cells/thymus; 5 months after
in3-ction of CD34~ Thy-l~ cells from a prim~ry culture, at
.,, . ~ ~
WO93/18137 PCT/USg3/0185
- 36 -
~3~36~ cells~thymus. Analogous results were observed for the
polyoma enhancer.
It is evident from the above results, that
pluripotent hematopoietic stem cells can be grown for
S extended periods of time, substantially expanded, in
culture, while retaining pluripotency. Thus, stem cells
may be obtained from a wide variety of source~, fetal or
adult, bone marrow or blood, and grown, so as to have an
expanded source of the cells. Furthermore, cells may be
infected with appropriate retroviruses, where the cells
will integrate the retroviral construct with functional
expression of a gene contained within the construct, where
in cells after differentiation and maturation, the gene is
retained and will be expressed.
lS All publications and patent applications mentioned in
this specification are herein incorporated by reference to
the same extent as if each individual publication or
patent application was specifically and individu~l~y
indicated to be incorporated by re~erence.
The invention now being fully descri~ed, it will be
~pparen~ to one of ordinary skill in the art that ~any
changes and modifications can bè ~ade thereto without
departing from the ~pirit or scope of the appended claims.
) 93/18137 21313 6 8 PCI`/US93/01852
-- 37 --
SEQUENCE LISTING
11) GENERAL INFORMATION:
(i) APPLICANT: SyStem;x, Inc.
(ii) TITLE OF INVENTION: Culturin~ of hematopo;etic stem cells
and thei- 9enetic engin~erino.
(iii) NUMBER OF SEQUENCES: 4
(iv~ CORRESPONDENCE ADDRESS:
~A) ADDRESSEE: Bertram 1. Rowland
(B) STREET: 4 Embarcadoro Center, Suite 3400
IC) CITY: San Francisco
(D) STATE: California
~E) COUNTRY: USA
~F~ ZIP: 94111
(v) COMPUTER READABLE FORM:
IA) MEDIUM TYPE: Floppv disk
(B) COMPUTER: IBM PC compatible
~C) OPERATING SYSTEM: PC-DOS/MS-DOS
D~ SOFTWARE: Patentln Release #1.0, Version #1.25
vfl CURRENT APPLICATION DATA:
A) :APPLICATION NUMBER: PCTIUS93/
(B~ FILING DATE: 03-MAR-1993
(C) CLASSIFICATION:
~viii) ATTORNEY/AGENT INFORMATION:
~A) NkME: Rowland, Bott am I
IB) REGISTRATION NUMBER: 20,015
C) REFERENCE/DOCKET NUMBFR: FP-55698/BIP~
lix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 781-1989
IB) TELEFAX: (415) 398-3249
t2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTEP~ISTICS:
(A) LENGTH: 21 base pairs
~B) TYPE: nucbic acid
~C) STRANDEDNESS: sin~le
(D) TOPOLOGY: linear
-tii) MOLECULETYPE: cDNA
xi) SEOUENCE DESCRIPTION: SEQ ID NO:1:
- ~ CATCGCATGA GCGAGCACGT A 21
'~ ~
.. . . .
WO 93/18137 PCI/USg3/0185~2"~
? ~3~ 12~ INFORMATIO~ FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 25 base pairs
~B) TYPE: nuchic acid
~C) STRANDEDNESS: single
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
Ixi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CGATGCCTGC TTGCCGAATA TCATG 25
12~ INFORMATION FOR SEQ ID NO:3:
li~ SEQUENCE CHARACTERISTICS:
A) LENGTH: 25 baso pairs
~B) TYPE: nucleic acid
~C~ STRANDEDNESS: single
~D) TOPOLOGY: linear
lii) MOLECULE TYPE: cDNA
:::
SEQUENCE DESCRIPTION: SEQ ID NO:3:
CTAGACTGGC CGTGCGACAT CCTCT 25
e) ~INFORMATION FOR SEQ ID NO:4: ''
SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
~B) TYPE: nuchic acid
~C) STRANDEDNESS: sin~le
~D) TOPOLOGY: lineu
~ii) MOLECULE TYPE: cDNA
~u) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CAATCATTAC TATGACAACA GTCTAG 26
, ~ ~
. .. . .. . . .. . . .. . .. .. .. .
