CA2131368A1 - Culturing of hematopoietic stem cells and their genetic engineering - Google Patents

Abstract

Human hematopoietic stem cells may be grown in longterm culture, particularly co-cultures with an appropriate stromal cell line, particularly in the presence of leukemia inhibitory factor, by itself or in conjuction with other factors. The cells are found to retain their function as demonstrated by an in vivo T-cell assay and their ability to form colonies of other lineages in methylcellulose. Transfection of the cells may be achieved with a virus, where improved results are obtained in the presence of leukemia inhibitory factor and optionally other hematopoietic factors.

Description

~ 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 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 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 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 (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 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 ::

'~'093/18137 PCT/US93/01852 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 ~'~93/18137 - PCT/US93/01852 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 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 .

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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).
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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

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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) . ~

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U~ o ~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.

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"'~93/18137 2 1 ~ 1 3 6 8 PCT/US93/01852 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 ~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 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:

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:

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:

, ~ ~

. .. . .. . . .. . . .. . .. .. .. .

Claims (20)

WHAT IS CLAIMED IS:

1. In a method for growing human hematopoietic stem cells in a culture medium, said stem cells being characterized by being CD34+ and Thy-l+, the improvement which comprises:
contacting said stem cells with a medium comprising at least about 10 ng/ml of leukemia inhibitory factor for at least 12 h; and growing said stem cells in a medium capable of supporting the growth of said stem cells.

2. A method according to Claim 1, wherein said leukemia inhibitory factor is present in a concentration of from about 2 to 20 ng/ml.

3. A method according to Claim 1, wherein said contacting is with at least one of IL-3, IL-6, GM-CSF, or steel factor in a concentration of at least 1 ng/ml.

4. A method according to Claim 1, wherein said growing is in the presence of medium conditioned by cells from a stromal cell line.

5. A method according to Claim 4, wherein said stromal cells are present in said medium.

6. In a method for growing human hematopoietic stem cells in a culture medium, said stem cells being characterized by being CD34+ and Thy-1+, the improvement which comprises:
contacting said stem cells with a medium comprising from about 2 to 20 ng/ml of leukemia inhibitory factor and at least one of IL-3, IL-6, GM-CSF, or steel factor in a concentration of at least 1 ng/ml for at least about 12 h;
and growing said stem cells in a medium capable of supporting the growth of said stem cells.

7. A method according to Claim 6, wherein said growing is in the presence of medium conditioned by cells from a stromal cell line.

8. A method according to Claim 7, wherein said stromal cells are present in said medium.

9. A method according to Claim 6, wherein said leukemia inhibitory factor is maintained in said growth supporting medium.

10. A method for transfecting human hematopoietic stem cells, said stem cells being characterized by being CD34+ and Thy-l+, the method comprising:

contacting human hematopoietic stem cells substantially free of dedicated cells with a retroviral vector tropic for human cells and comprising a DNA
composition of matter which comprises a DNA element which mediates transcription in human hematopoietic cells, said contacting being in a medium comprising at least 10 ng/ml of leukemia inhibitory factor for a time sufficient for said virus to enter said stem cells.

11. A method according to Claim 10, wherein said medium further comprises at least one of IL-3, IL-6, GM-CSF, or steel factor in a concentration of at least 1 ng/ml.

.

12. A method according to Claim 10, wherein said DNA
composition of matter is a gene transcriptionally active in human hematopoietic cells.

13. A method according to Claim 10, further comprising growing said hematopoietic stem cells comprising said DNA composition of matter in culture in a medium capable of supporting the growth of stem cells.

14. A method according to Claim 13, wherein said growth supporting medium is a medium conditioned by cells from a stromal cell line.

15. A method according to Claim 14, wherein said stromal cells are present in said growth supporting medium.

16. A method according to Claim 15, wherein said leukemia inhibitory factor is maintained in said growth supporting medium.

17. A human hematopoietic stem cell composition, said stem cells being characterized by being CD34+ and Thy-1+, and substantially free of dedicated hematopoietic cells, comprising a DNA composition of matter resulting from integration of said DNA composition of matter.

18. A human hematopoietic stem cell according to Claim 17, wherein said DNA composition of matter is a gene trancriptionally active in a hematopoietic cell.

19. A human hematopoietic stem cell according to Claim 18, wherein said gene is a G418 resistance gene.

20. A human hematopoietic cell descended from an human hematopoietic stem cell according to Claim 17.