Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ISOLATION OF ~AMMAT-TAN HEMATOPOIETIC STEM CELLS
.
~2~r~T~n AppTTCATIONS
This application is a Continuation-in-Part of
cor~n~;ng U.S. Patent Application Serial No. 08/47:L,758,
filed June 6, 1995, the entire teachings of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
Hematopoietic stem cells (HSC) are multipotent:ial
cells which reside in the bone marrow and replenish all
adult hematopoietic lineages through the lifetime of an
animal. Lack of a purified population of stem cells has
hampered definitive unders~n~in~ of the factors which
regulate their growth and differentiation. Several schemes
for the enrichment of hematopoietic stem cells from murine
bone marrow have been developed, but a phenotypica]!ly
homogeneous population of stem cells has not been
identified to date (Visser, J.W., et al., J. Exp. ~ed.,
159:1576-90 (1984); Pallavicini, M.G., et al., Exp~
Hematol., 13:1173-81 (1985); Spangrude, G.J., et al.,
Science, 241:58-62 (1988); Szilvassy, S.J., et al., Blood,
74:930-9 (1989); Jones, R.J., et al., Nature, 347:188-9
(1990); Nicola, N.A., et al., Blood, 58:376-86 (1981);
Bertoncello, L., et al., Exp. ~ematol., 13:999-1006
(1985)).
The ability to obtain a phenotypically homogeneous
population of stem cells is important in achieving greater
success with bone marrow transplants (BMTs). For example,
autologous BMT could become a candidate for use in leukemic
~ and other cancer therapies if it were possible to
definitively remove cancerous cells from the bone marrow.
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Isolation of pure reconstituting HSCs may allow this.
Furthermore, non-autologous BMT may also benefit from HSC
purification, as transplantation of purified HSCs may
ameliorate graft versus host disease (GVH) syndromes. In
addition, a homogeneous stem cell population would be
advantageous for hematopoietic stem cell gene therapy.
Previous attempts to infect unmanipulated murine and human
HSCs with recombinant retroviruses have met with mixed
success, presumably at least in part due to the inability
of retroviral vectors to integrate into ~uiescent HSCs,
which are widely held to constitute the majority of the HSC
population. Thus, purification of HSCs and the ability to
distinguish between quiescent HSCs and cycling HSCs would
have important implications for HSC gene therapy.
S~MMARY OF THE INVENTION
The present invention is based on the discovery that
unusual uptake and fluorescence properties of a dye in
hematopoietic stem cells tHSCs) make it possible to purify
HSCs and isolate a subpopulation of HSCs that are naturally
replicating in vivo. Thus, the invention relates to a
method of purifying mammalian hematopoietic stem cells from
a bone marrow cell population.
~ n one embodiment, a bone marrow cell sample is
obtained and combined with a fluorescent, lipophilic vital
dye which is a substrate for a multiple drug resistant
protein (i.e., mdr protein) under conditions appropriate
for cells to take up the dye. The term "substrate" is
defined herein as a substance which is removed by mdr. The
resulting combination is exposed to an excitation
wavelength which results in fluorescence of the dye, which
is measured using an emission wavelength. The amount of
dye contained by each population of cells resolved with the
emission wavelength is determined. The population of
-
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nucleated cells which contains the smallest amount: of dye
~t the emission wavelength is purified, mammalian HSCs.
In another embodiment a bone marrow cell sample is
obtained and combined with a fluorescent, lipophi].ic vital
dye which is a substrate for a mdr protein, under
conditions appropriate for cells to take up the dye. The
resulting combination is exposed to an excitation
wavelength which results in fluorescence of the dye, which
is measured using two emission wavelengths simultaneously.
The amount of dye contained by each population of cells
resolved with the two emission wavelengths is determined.
The population of nucleated cells which contains the
smallest amount of dye at both wavelengths is purified,
~ lian HSCs.
The present invention also relates to a method of
separating cycling HSCs from ~uiescent HSCs in a purified
mammalian HSC sample. In the method, a purified HSC sample
is obtained and combined with a fluorescent, lipophilic
vital dye which binds DNA and is a substrate for a mdr
protein and an inhibitor of the mdr protein under
conditions appropriate for the cells to take up the dye and
the inhibitor. The amount of dye present in the purified
HSCs is measured, wherein purified HSCs which contain the
smallest amount of dye have a 2n quantity of DNA (i.e.,
which refers to the base number of chromosomes in a cell)
and are designated as ~uiescent HSCs, and purified HSCs
which contain a greater amount of dye have a greater amount
of DNA (i.e. >2n) and are designated cycling ~SCs. This
would also apply to obtaining cycling cells from a
population of cells having high mdr activity. For example,
a heterogeneous population of cells such as bone marrow can
be combined with the dye and the inhibitor. Cycling and
non-cycling populations are determined as describe~ above.
In this case a heterogenous population of cycling cells are
isolated and can be used for infecting with retroviruses.
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Further, the invention relates to a method of
identifying an agent which is a fluorescent, vital dye
which is lipophilic for use in purifying mammalian HSCs.
In one embodiment, a bone marrow cell sample is obtained
and combined with the agent under conditions appropriate
for the cells to take up the agent. The resulting
combination is exposed to an excitation wavelength which
results in fluorescence of the agent, which is measured
using an emission wavelength and the amount of dye
exhibited by each population of cells is determined. If
distinct populations of nucleated cells are observed in
which one of the population of cells contains the smallest
amount of dye and the population is purified, mammalian
HSCs, then the agent is a fluorescent vital dye which is
lipophilic for use in purifying HSCS.
In another embodiment, a bone marrow cell sample is
obtained and combined with the agent under conditions
appropriate for the cells to take up the agent. The
resulting combination is exposed to an excitation
wavelength which results in fluorescence of the agent which
is measured using two emission wavelengths simultaneously.
The amount of dye contained by each population of cells
resolved with the two emission wavelengths is determined.
If distinct populations of nucleated cells are observed in
which one of the population of cells contains the smallest
amount of dye and the population is purified, mammalian
HSCS, then the agent is a fluorescent, vital dye which is
lipophilic for use in purifying HSCs.
The present invention also relates to a method of
transplanting bone marrow in a mammalian host, such as a
human, comprising introducing into the host the purified
HSCs described herein. The invention further relates to a
method of n vivo administration of a protein comprising
infecting or transfecting a purified HSC with a construct
comprising DNA or RNA which encodes a protein of interest
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and introducing the infected or transfected purified HSC
into the host, in which the protein of interest i~
expressed. In a particular embodiment, exogenous DNA
encoding a protein of interest is infected or transfected
into a cycling HSC.
The present invention also relates to hematopoietic
stem cells purified using or obtaina~le by (obt~;ne~ by)
the methods described herein. In one embodiment, the
purified HSCs of the present invention are CD34~. In
another embodiment, the purified HSCs are side population
(SP) cells. In another embodiment, the purified HSCs have
at least one of the following characteristics: glycophorin
A~, CD38~tn~, CD13n~, CD15~, CD19n~ and CD20n~.
Thus, the present invention allows for the use of
purified HSCs in bone marrow transplants. In addition, the
method of separating cycling HSCs from quiescent HSCs
provides a means of obtaining cycling HSCs, separated from
quiescent HSCs for transfection with a construct encoding a
protein of interest. As discussed above, recombinant
retroviral are unable to integrate into quiescent i~SCs.
Thus, the ability to specifically transfect cycling HSCs
will provide greater success in in vivo administration of
proteins to or by HSCs.
BRIEF DESCRIPTION OF THE FIGURES
Figure lA is a graph of Hoechst red versus blue
fluorescence on a linear scale of whole murine norrnal bone
marrow stained with Hoechst 33342 stain, in which t:he boxed
region represents the hematopoietic stem cell activity,
which is 0.1% of the total bone marrow cell populat:ion.
Figure lB is a graph illustrating the analysis of Sca-
1 and lineage marker staining of whole murine normal bone
marrow.
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Figure lC is a graph of Hoechst red versus blue
fluorescence on a linear scale of whole murine normal bone
marrow stained with Hoechst 33342 stain, in which the
region indicated in Figure lB is used as a live gate in
conjunction with the gate to exclude red and dead cells.
Figure lD is a graph illustrating the analysis of Sca-
1 and lineage marker st~; n; ng of whole murine normal bone
marrow cells which fall exclusively into the boxed region
indicated in Figure lA.
Figure lE is a graph of Hoechst red versus blue
fluorescence on a linear scale of Sca-l enriched murine
normal bone marrow stained with Hoechst 33342 stain, in
which the specific side region indicated in Table 2 is
delineated.
Figure 2A is a graph of Hoechst red versus blue
fluorescence on a linear scale of whole bone marrow stained
with Hoechst 33342.
Figure 2B is a graph of Hoechst red versus blue
fluorescence on a linear scale of whole bone marrow stained
with Hoechst 33342 in the presence of Verapamil.
Figure 3A is a graph of Hoechst red versus blue
fluorescence on a linear scale of purified HSCs restained
with Hoechst 33342 in the presence of verapamil.
Figure 3B is a graph of propidium iodide (PI) versus
cell number of purified HSC stained with PI.
Figure 4A is a graph of Hoechst red versus blue
fluorescence on a linear scale of human whole bone marrow
cells stained with Hoescht 33342 stain.
Figure 4B is a graph of Hoechst red versus blue
fluorescence on a linear scale of human cord blood cells
stained with Hoescht 33342 stain.
Figure 5A is a graph of Hoechst red versus blue
fluorescence on a linear scale of porcine bone marrow cells
stained with Hoescht 33342 stain.
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Figure 5B is a graph of Hoechst red versus blue
fluorescence on a linear scale of Rhesus bone marrow cells
stained with Hoescht 33342 stain.
D~TAILED DESCRIPTION OF THE INVENTION
The invention relates to a method of purifyi~g
mammalian hematopoietic stem cells (HSCs) from a bone
marrow cell population.
In one embodiment, a bone marrow cell sample is
obtained and combined with a fluorescent, lipophilic vital
dye which is a substrate for a multiple drug resistant
protein (i.e., mdr protein) under conditions appropriate
for cells to take up the dye. The term "substrate" is
defined herein as a substance which is removed from a cell
by mdr. The resulting combination is exposed to an
excitation wavelength which results in fluorescence of the
dye, which is measured at an emission wavelength and the
amount of dye exhibited by each population of cells is
determined. The population of nucleated cells which
contains the smallest amount of dye at the emiSSiolQ
wavelength is purified, mammalian HSCs.
In another embodiment, a bone marrow cell sample is
obtained and combined with a fluorescent, lipophil:ic vital
dye which is a substrate for a mdr protein, under
conditions appropriate for cells to take up the dye. The
resulting combination is exposed to an excitation
wavelength which results in fluorescence of the dye, which
is measured using two emission wavelengths simultaneously.
The amount of dye contained by each population of cells
resolved with the two emission wavelengths is determined.
The population of nucleated cells which contains the
smallest amount of dye at both wavelengths is purified,
mammalian HSCs. As described herein, the method of
purifying HSCs has been used to purify side population (SP)
cells from four types of mammals: murine, human, monkey and
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porcine. Characterization of the murine SP cells indicate
they are purified HSCs. Further characterization of the
human, monkey and porcine SP cells will confirm they are
purified HSCs. Applicants have shown that the purified
HSCs described herein successfully repopulated stem cell
activity in lethally irradicated recipients, providing 1000
fold enrichment of HSC activity in mouse bone marrow. In
addition, the HSCs of the present invention have been shown
to allow survival of lethally irradiated recipients.
Thus, the present invention relates to a method for
the isolation or purification of mammalian HSCs, and is
based on the use of FACS analysis of bone marrow cells
stained with a fluorescent vital dye. Starting with
untreated bone marrow, 1,000 fold enrichment of in vivo HSC
reconstitution activity can be consistently achieved. The
purification strategy has also led to a method for
separating cycling from quiescent stem cells, which will
facilitate efficient gene transfer into hematopoietic stem
cells.
The dye which can be used in the method for purifying
HSCs is a fluorescent, vital dye which is a substrate for a
mdr protein. Preferably, the dye is also lipophilic. In
the method of separating cycling HSCs from quiescent HSCs,
the dye must also be a DNA binding dye. In some instances,
one dye can be used in the method for purifying HSCs and in
the method for separating cycling HSCs from quiescent HSCs.
For example, as described in Examples 1 and 3, Hoechst
33342 dye (Hoechst dye) can be used in both the method for
purifying HSCs and the method of separating cycling HSCs
from quiescent HSCs. Although the method of the present
invention is exemplified using Hoechst dye (Example 1),
other dyes can be used to practice the methods of the
present invention. For example, Rhodamine 123 might be
used to practice the invention, particularly in the
embodiment in which purified HSCs are isolated.
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Hoechst 33342 is a fluorescent DNA-binding dye useful
for flow cytometry analysi~. The dye is readily t:aken up
by live cells, in which it binds to DNA. The quantity of
Hoechst 33342 fluorescence relates to DNA content in a cell
~ 5 and, therefore, is an indicator of cell cycle. Howe~er, as
described in Example 1, when Hoechst dye was used to
~YA~; ne ~DNA content in murine bone marrow cells by st~n~Ard
methods, a complex fluorescence pattern was observed. When
dye fluorescence was observed simultaneously at two
emission wavelengths (the red and the blue), several
distinct populations could be resolved. These populations
are shiflted relative to each other predominately ~ith
regard to fluorescence on the Hoechst "red" axis (Fig. lA).
Analysis of hematopoietic cells in this staining profile
indicated that one population of cells, boxed in Figure lA,
was predominately Sca-l+lin~ (Fig. lD). That is, 75% of
the cells from the boxed region in Fig. lA expressed Ly-
6A.2 (Sca-l+), and a low level of six lineage antigens
(detectecl by a cocktail of antibodies against B220, Gr-1,
Mac-1, Cl)4, CD5, and CD8). These cell surface
characteristics have previously been identified on murine
hematopoiLetic stem cells by Applicants and others
(Spangrucle, G.J., et al., Science, 241:58-62 (1988);
Uchida, ~. and Weissman, I.L., J. Exp. Med., 175:175-84
(1992)). Characterization of the purified human SP
describecl herein indicate that the purified HSCs are:
glycopho~in An'g, CD38l~'n'g, CD13n~, CD15n~, CDl9n~, CD20nCg, and
CD34n'g. ICharacterization of purified HSCs in mouse
indicate that the purified mouse HSCs are: c-kit~S, Sca-1~s,
Gr-lD'g"~, Mac-ln~'~, CD4n~"~, CD8n'~, B220n~, CD5n~, CD43~,
CD45~s, Ml69~s, AA4n~/l~, class I MHC~s, class II MHCn~,
Rhodamine 123~, and Wheat germ agglutinin (WGA)~s. This
population, referred to as the side population, or SP,
represents 0.1% of total bone marrow. These SP cells were
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also a sub-population (approximately 5%) of the total Sca-
l+,lin~ cells in the bone marrow.
To examine the functional properties of these cells,
long term bone marrow transplantation experiments were
performed, as described in Example 2. A competitive
repopulation assay, in which defined numbers of purified
cells (i.e., SP cells) were transplanted into lethally
irradiated recipients along with unfractionated but
distinguishable whole bone marrow cells (i.e.,
unfractionated cells), was used. In this semi-quantitative
method, the relative stem cell activity of sorted or SP
cells versus unfractionated cells is assessed by
determining the percentage of peripheral blood cells
descended from each of the two input populations in the
bone marrow transplant recipients (Harrison, D.E., Blood,
55:77-81 (1980); Harrison, D.E., et al., Proc. Natl. Acad.
sci . U.S.A., 85: 822-826 (1988)). It is generally accepted
that at four months post-transplant, the majority of
peripheral blood cells are derived from long term
reconstituting stem cells introduced in the transplant
instead of from committed progenitors. In addition,
differentiated cells and progenitors in the unfractionated
competitor bone marrow rescue recipients from the otherwise
lethal irradiation. This allows the long term multi-
lineage repopulating functions of the input HSC to beexamined separately from short-term activities.
As described in Example 2, in competitive repopulation
assays with the SP cells, average enrichments of HSC
activity of approximately l,000 fold over multiple
experiments were achieved (Table 1). Since the SP cells
represent 0.1% of the total bone marrow, this indicates
that most if not all reconstituting cells in normal mouse
bone marrow reside in the SP fraction. Moreover,
competitive repopulation experiments with other Hoechst-
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stained populations shown in Fig. lA, revealed al~nost no
additional stem cell activity. See Table 2.
Having established by competitive repopulation that
stem cell activity fell into the SP region, the ability of
~ 5 that population of cells to protect recipients from lethal
irradiation (i.e., radioprotection) was investiga1:ed. As
shown by data from three separate experiments in Table 3,
approximately 150 purified cells protected 50% of the
recipients from lethal irradiation. That is, the presence
of approximately 150 purified cells in the lethally
irradiated recipients resulted in survival of 50~ of the
recipients.
It was of interest to account for the unusual Hoechst-
staining properties of the hematopoietic stem cells.
lS Since it is known that several vital dyes are actively
pumped out of the cells by the mdr protein (i.e.,
p-glycoprotein) (Chaudhary, P.M. and Roninson, L.E~., Cell,
66:85-94 tl99l)), the possibility that the exceptionally
low Hoechst fluorescence was due to higher mdr act:ivity in
murine hematopoietic stem cells was investigated. As
described in Example 3, bone marrow stained with Hoechst
33342 in the absence or presence of the drug verapamil, an
inhibitor of mdr, demonstrates that the stem cell
population is visible (i.e., able to be isolated) in the
absence of verapamil (Fig. 2A, Fig. 2B).
Based upon the ability of verapamil to block the
efflux of Hoechst dye, it was postulated that separation of
replicating stem cells from quiescent stem cells is
possible. As discovered and described herein, this has
been shown to be correct, and can further be used to
separate replicating stem cells from quiescent stem cells.
That is, as shown herein, in the presence of verapamil, the
efflux activity of Hoechst dye is blocked, and a higher
concentration of Hoechst dye is retained in the cells. In
cell populations which do not contain high amounts of mdr
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activity, Hoechst dye reflects DNA content and has commonly
been used in cell cycle analysis. Cycling cells have a
greater amount of DNA than quiescent cells. Thus, a
greater amount of dye will be present in cycling HSCs
resulting in greater fluorescence of the dye present in the
cycling HSCs when the dye is excited at the appropriate
wavelength. Conversely, a lower amount of dye will be
present in quiescent cells resulting in lower fluorescence
of the dye present in quiescent HSCs when the dye is
excited at the appropriate wavelength. Thus, measuring the
relative amount of fluorescence of a dye in purified HSCs
allows for separation of cycling HSCs from quiescent HSCs
(Technigues in Cell Cycle Analysis, ed. Gray, J.W. and
Darzynkiewicz, Humana Press, Clifton, NJ (1987)).
Accordingly, as described in Example 4, HSCs were
purified (sorted) on the basis of low Hoechst staining, to
produce SP cells as described herein. The sorted or SP
cells were restained with Hoechst dye in the presence of
verapamil. Cell-cycle analysis was performed as described
in Example 1 and the number of stem cells in S-G2M (i.e.,
cycling HSCs) was found to range between 1 and 3% of the
purified cells (Fig. 3A). This figure correlates well with
the number shown to be in S-G2M by propidium iodide
staining of the purified cells (Fig. 3B). Stem cells in
Go~GI (i.e., quiescent HSCs) made up the rest of the
purified cells.
With a view to using cycling stem cells as targets for
gene transfer, such as retroviral-mediated gene transfer,
the relative engraftment potential of stem cells that were
in Go~GI versus S-G2M was compared. These subsets of the SP
population were purified by flow cytometry using the
verapamil-blocking strategy for cell cycle analysis
described in Example 1, and tested in the competitive
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repopulation assay described in Example 2. Table 4 shows
the results from the experiment.
Thus, the present invention also relates to ~ method
of separating cycling HSCs from quiescent HSCs in a
S purified stem cell sample. In this method, a purified HSC
sample is combined with two substances: a fluoresc:ent,
lipophilic vital dye which binds DNA and is a substrate for
a mdr protein and an inhibitor of the mdr protein. This is
carried out under conditions appropriate for the cells to
take up the dye and the inhibitor. The amount of dye
present in the HSCs is subsequently measured or observed
for diff~erences in intensity of fluorescence. The extent
to which the dye is contained by a cell is in proportion to
the quantity or concentration of DNA in the cell.
Quiescent ~SCs contain less or a lower concentration of,
DNA than do cycling HSCs and, therefore, quiescent cells
take up less dye than is taken up by cycling HSCs.
Quiescen1t HSCs can be distinguished from (i.e., separated
from) cycling HSCs on the basis of the quantity or
concentration of the vital dye present. Those which
contain .~ lesser quantity or concentration of dye are
quiescenlt HSCs and those which contain a larger quantity or
concentration of dye are cycling HSCs. This would also
apply to obtaining cycling cells from a population of cells
having high mdr activity. For example, a heterogeneous
population of cells such as bone marrow can be combined
with the dye and the inhibitor. Cycling and non-cycling
populations are determined as described above. In this
case a heterogenous population of cycling cells are
isolated and can be used for infecting with retroviruses.
The invention further relates to a method of
identifying an agent which is a fluorescent, vital dye for
use in purifying hematopoietic stem cells. In one
embodiment, a bone marrow cell sample is obtained and
combined with the agent under conditions appropriate for
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the cells to take up the agent. The resulting combination
is exposed to an excitation wavelength which results in
fluorescent of the agent, which is measured using an
emission wavelength. The amount of dye contained by each
population of cells resolved with the emission wavelength
is determined. If distinct populations of nucleated cells
are observed in which one of the population of cells
contains the smallest amount of dye and the population is
purified, mammalian HSCs, then the agent is a fluorescent
vital dye which can be used in purifying hematopoietic stem
cells.
In another embodiment, an agent is identified by
combining a bone marrow cell sample and an agent to be
assessed, under conditions appropriate for the cells to
take up the agent. The resulting combination is exposed to
an excitation wavelength which results in fluorescence of
the dye. The dye fluorescence is measured using two
emission wavelengths simultaneously, so that distinct
populations of live bone marrow cells are resolved on the
basis of fluorescence. Dye uptake is assessed in each
population of cells. If distinct populations of cells are
observed in which one of the population of cells contains
the least amount of dye at both wavelengths and the
population is purified, mammalian HSCs, then the agent is a
fluorescent, vital dye which is for use in purifying HSCs.
The stem cell purification strategy described herein
can be used with any suitable mammalian (e.g., vertebrate)
species. Applicants have shown that SP cells can be
purified from four types of mammals: mice, humans, monkeys
and pigs. As described in Example 5, adult human bone
marrow cells and human cord blood cells have been stained
with Hoechst 33342 and a staining pattern remarkably
similar to that which was observed for murine cells was
observed for these cells. The frequency of cells in the
human side population is also approximately 0.03% (range
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0.01% to 0.05%). As described in Example 6, HSCs from
porcine bone marrow and monkey bone marrow were p~lrified by
~ using the method of the present invention. Further
characterization of the HSCs purified from monkey~;, which
~ 5 is described in Example 7, indicates that greater stem cell
activity is in the SP cells which are CD34~.
Obtaining a bone marrow cell sample from a maLmmal for
use in tlhe methods of the present invention can b~ achieved
using rolutine methods known to those of skill in the art.
For exam]ple, as described in Example 1, the bone marrow
cells ca]n be obtained by extracting a cell suspension from
the femurs and/or tibias of the mammal, passing the cells
through an orifice (e.g., an 18 gauge needle) and pelleting
the cell~s by centrifugation.
The HSCs for use in the methods of the present
inventio~ can be obtained from any suitable mammalian
source sllch as rodent (e.g., rats, mice), primate, dog,
pig, cat, monkey, and/or human sources.
In lthe method of separating cycling and quiescent
purified HSCs, the purified HSCs can be obtained using any
suitable method for purifying HSCs. For example, the
method for purifying HSCs as described herein can be used.
Alternatively, a method of purifying HSCs based on cell
surface markers can be used (Visser, J.W., et al., ~. Exp.
Med., 1~'3:1576-90 (1984); Pallavicini, M.G., et al., Exp.
Hematol., 13:1173-81 (1985); Spangrude, G.J., et al.,
Science, 241:58-62 (1988); Szilvassy, S.J., et al., Blood,
74:930-9 (1989); Jones, R.J., et al., Nature, 347:188-9
(1990); Nicola, N.A., et al., Blood, 58:376-86 (1981);
Bertonce]Llo, L., et al., ~xp. ~ematol., 13:999-1006
(1985)).
The excitation wavelength used in the method o~
purifying HSCs, is a suitable wavelength which will excite
the particular dye chosen to a measurable extent. For
=
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example, in the embodiment in which the Hoechst dye is used
to purify HSCs, an appropriate excitation wavelength is
from about 250 nm to about 450 nm, and in a particular
embodiment, is about 350 nm. Hoechst dye emission can be
detected at a range of wavelengths, from about 400 nm to
about 700 nm, and in a particular embodiment, about 600 nm.
In another embodiment about 450 nm and about 650 nm can be
used simultaneously to detect Hoechst dye emission.
In the method of purifying HSCs, the fluorescence of
the dye can be measured at one emission wavelength.
Alternatively, two emission wavelengths can be used
simultaneously to measure fluorescence of the dye.
Suitable emission wavelengths are those which will measure
the fluorescence of the dye chosen so that distinct
populations of live bone marrow cells are resolved. For
example, in the embodiment in which the Hoechst dye is used
to purify HSCs, the fluorescence of the Hoechst dye is
measured at two wavelengths using a 450 band pass (BP) 20
and a 675 edge filter long pass (EFLP) optical filter. As
indicated by the graph of Figure lA, the fluorescence of
the Hoechst dye can also be measured using only the red
emission wavelength (i.e., 675 nm) to obtain purified HSCs.
This was a surprising result since normally 450 nm emission
wavelength is used with Hoechst dye because that is its
peak emission wavelength.
The amount of dye used in the methods of the present
invention functionally will generally be from about l ~g/ml
to about 20 ~g/ml dye, preferably from about 5 ~g/ml to
about 15 ~g/ml dye and in particular, about 5 ~g/ml dye.
The staining time with the dye (i.e., the length of
time cells are exposed to dye) varies depending on the
temperature at which staining is to occur and the dye
concentration in the methods of the present invention.
Thus, staining can occur overnight or over a number of days
at the appropriate temperature. In particular, the
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st~;n;ng time with the dye can be from about 30 minutes to
about l80 minutes, preferably between about 60 minutes to
about 12l~ minutes. In a particular embodiment of the
method for purifying HSCs, the staining time with the
Hoechst dye is about 90 minutes. In a particular
emhoA;me~lt of the method for separating cycling and
quiescen1t purified HSCs, the stA;n;ng time with the Hoechst
dye is about 60 minutes.
The temperature at which stA; n;ng with the dye can be
carried out is from about 4~C to about 45~C, preferably
about 15''C to about 45~C, and in particular, about 37~C in
the methods of the present invention. The temperature at
which staining with the dye can be carried out can also be
room temperature (i.e., about 25~C).
In a particular embodiment of the present method of
purifyin~ HSCs, 5 ~g ~oechst dye is used to stain a bone
marrow ce~ll population and, thus, the HSCs it contains, for
90 minutes at 37~C. In a particular embodiment for the
method of separating cycling HSCs from quiescent HSCs, lO
~g Hoech~;t dye is used to stain the murine bone marrow cell
population for 60 minutes at 37~C. In another embodiment,
human and monkey bone marrow were stained for 120 minutes
at 37~C. In a further embodiment, porcine bone marrow was
stained i-or 90 minutes at 37~C.
In 1:he method of separating cycling HSCs from
quiescen1: HSCs, an inhibitor of the mdr protein is a
substance or agent which interferes with the activity of
the mdr protein in the HSCs. That is, an inhibitor of the
mdr protein is a substance or agent which interferes with
the abiliity of the mdr protein to remove the dye from the
HSCs. Inhibitors of the mdr protein include verapamil,
antibodies directed against mdr (i.e., anti-multiple drug
resistant: protein antibody), reserpine, PAK-104P,
vincristine and SDZ PSC 833. The term "multiple drug
resistant: protein" as used herein includes the multiple
CA 02223492 1997-12-04
W O 96~9489 . PCTrUS96/09415
-18-
drug resistant (mdr) protein and proteins which exhibit
mdr-like activity (i.e., an mdr-like efflux of a dye from a
HSC). For example, analogs or derivatives of the mdr
protein, are included in the term "multiple drug resistant
protein".
In the method of separating cycling HSCs from
quiescent HSCs, a low amount or quantity of DNA (i.e., 2n
DNA or base number of chromosomes in a cell) in HSCs
indicates the presence of quiescent HSCs (i.e., HSCs cells
in the Go~GI phase) and a high amount or quantity of DNA
(i.e., >2n DNA) in HSCs indicates the presence of cycling
HSCs (i.e., HSCs cell in the S-G2M phase).
The HSCs obtained by the method of the present
invention can be used in a variety of ways. For example,
lS the HSCs of the present invention can be transplanted into
a host (e.g., mammal, particularly human) in need of a bone
marrow transplant (e.g., an irradiated host or a host
undergoing chemotherapy). In addition, the HSCs of the
present invention can be used to treat diseases or
conditions in which an individual needs bone marrow cells.
The present invention further relates to a method of
providing a host with purified HSCs comprising the step of
introducing into the host the purified HSCs described
herein. In particular embodiments, cycling HSCs or
quiescent HSCs are introduced into the host.
The methods of the present invention can be used for
n vivo administration of protein by transfecting or
infecting purified HSCs with recombinant vectors or
constructs comprising DNA which encodes a protein of
interest. In particular embodiment, cycling or quiescent
purified HSCs are transfected or infected with recombinant
constructs comprising DNA or RNA which encodes a protein of
interest. Previous attempts to infect unmanipulated HSCs
with recombinant retroviral vectors have met with mixed
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. .. .
-i9-
success, presumably at least in part due to the inability
of vectors to integrate into ~uiescent HSCs, which as shown
herein constitute the majority of the HSCs populat:ion.
Thus, thl~ method of separating cycling HSCs from g[uiescent
HSCs allows for greater success in infecting HSCs with
recombinant retroviral vectors. The "administration of
protein" by definition includes the delivery of a
recombinant RSC which expresses a protein n vivo. For
example, a purified cycling HSC containing a vector,
wherein l:he vector contains a DNA or RNA sequence w~ich
expresse-; a protein of interest can be administered to a
host under conditions in which the protein of interest is
expresse~l in vivo (see e.g., United States patent Number
5,399,346 which is herein incorporated by reference).
Weissman, et al. have worked with Sca~ Ve, li~e,
and Thy-l~ cells, to characterize murine hematopoietic
stem cel]s. However, it has become increasingly clear that
this population is not homogeneous for stem cell activity
(Li, C.L. and Johnson, G.R., "Rhodamine 123 reveal~s
heterogeneity within murine Lin-, Sca-1+ hemopoietic stem
cells", J. Exp. Med. 175:1443-1447 (1992); Spangrude, G.J.
and Johns,on, G.R., "Resting and activated subsets of mouse
multipotelnt hematopoietic stem cells", P~oc. Natl. Acad
sci. USA 87:7433-7437 (1990)). These cells can be
subfractionated with the use of the vital dye Rhodamine-
123, where the 5-10% of cells that contain the lowest
amount of the dye after staining have most if not all of
the long-term-reconstituting activity of the population.
Fleming et al., ~. Cell. Biol., 122:897-902 (~993)
have compared the reconstitution potentials of Hoechst
33342-sorted Go~GI and S-G2M fractions of Thyl.1'~Lin--'~ Sca-
l+ stem cells. They observed 18% of the cells in S-G2M,
and reported a lower reconstitution capacity of this sub-
set of cells. However, as shown here, Hoechst fluorescence
CA 02223492 1997-12-04
W O 96~9489 PCT~US96/09415
-20-
in HSC reflects mdr-like efflux activity, not DNA content,
unless the efflux activity is blocked with inhibitors such
as verapamil. Therefore, the Hoechst content of their HSC
would be lower than other cells in their population, and
even cycling HSC would fall into their Go~GI peak (and
perhaps below it). Therefore, the reconstitution potential
of the cycling fraction was probably underestimated. They
also ~Y~r;ne the cell cycle profile of the Rhl23~ sub-set
of Thyl.1~ Lin-/bSca-1+ cells by propidium iodide staining,
and find approximately 3% of these cells are in S-G2M.
This figure correlates well with the number of our SP cells
in S-G2M, and it is expected that these populations are
qualitatively extremely similar.
The ability to obtain populations of cycling stem
cells also has important implications for hematopoietic
stem cell therapy, including administration of the cells,
as obtained by the present method as modified, such as by
the introduction of an exogenous gene encoding a protein of
interest (e.g., a therapeutic protein). Previous attempts
to infect unmanipulated murine and human HSCs with
recombinant retroviruses have met with mixed success,
presumably at least in part due to the inability of
retroviral vectors to integrate in quiescent cells. As
shown here, only 1-3% of HSC are in cycle. The
purification of the rare population of stem cells that are
naturally replicating in vivo provides an alternative to
transduction strategies based on attempts to influence the
cell cycle status of stem cells.
The invention is further illustrated in the following
examples.
~ CA 02223492 1997-12-04
WC~ 9C~9489 I'CT/U596~0941~;
.
~-- ---EXEMPLIFICATION
~x~mple 1: Characterization of Hoechst 33342 fluorescence
~n whole murine bone marrow.
Preparation of Hoechst 33342-stained Murine Bone ~arrow
Murine bone marrow was extracted from the femurs and
tibias of C57B1/6 mice, a single cell suspension was made
by passage of the bone marrow through an 18 gauge needle,
and the cells were pelleted by centrifugation. The bone
marrow c,ells were resuspended at lo6 cells per ml in pre-
warmed D]~EM contain 2% fetal calf serum, lmM HEPES, 50
units/ml Penicillin, 50 ~g/ml Streptomycin, and 5~g per ml
Hoechst 33342 (Sigma) and incubated for 90 minutes at 37~C.
The resolution of these populations is sensitive to the
staining time and the Hoechst dye concentration (Elwart,
J.W. and Dormer, P., Cytometry, 11:239 - 43 (1990)).
After Hoechst staining, cells are maintained at 4~C
until FACS analysis. Any antibody st~; n; ng or manipulation
is performed at 4~C following the Hoechst stain. After the
final manipulation, the bone marrow cells were resuspended
in HBSS+ containing 2~g/ml propidium iodide (PI). The
addition of PI did not affect the Hoechst staining profile,
but allowed exclusion of dead cells as described below.
- i. '
Flow Cytometer Set-Up
Ana:Lysis and sorting were performed on a dual laser
FACStar-plus or Facsvantage (Becton Dickenson). T~he
Hoechst clye was excited by the first Argon laser at 350nm
and its fluorescence was measured at two wavelengths using
a 450 band pass (BP) 20 and a 675 edge filter long pass
(EFLP) oE)tical filter (Omega Optical, Brattleboro ~T). A
610 dichroic mirror short pass (DMSP) was used to separate
the emission wavelengths. A 640 EFLP with 640 DMLP have
. ~
CA 02223492 1997-12-04
W O 96~9489 PCTAJS96/09415
-22-
also been used with an Enterprise laser (Coherent).
Propidium iodide (PI) fluorescence was also measured
through the 675 EFLP (having been excited at 350 nm).
Hoechst "blue" represents the 450 BP filter, the st~n~rd
analysis wavelength for Hoechst 33342 DNA content analysis.
Cells positive for PI were seen on the far right of the
Hoechst "red" (675 EFLP) axis shown, and excluded. Both
Hoechst blue and red fluorescence are shown on a linear
scale. Optimal cvs off of the first laser are necessary to
finely resolve the stem cell population. The second Argon
laser at 488 nm was used to excite standard fluorochromes
(e.g., fluorescein or phycoerythrin if necessary). No
cross compensation was necessary. The gating on forward
and side scatter was not stringent, only erythrocytes and
debris were excluded. Re-analysis of sorted populations
showed purity greater than 98%.
Flow cYtometry Profile: Identification of the Side
Population (SP) Cells
The Hoechst 33342-stained murine bone marrow is placed
on the flow cytometer. Since the Hoechst fluorescence is
analyzed on a linear scale, optimal cvs are obtained with a
relatively low sample differential, but if the cells are
resuspended at a sufficiently high concentration, they may
still be run at 3000-5000 cells per second. The sample can
be maintained at 4~C. Initially, the voltages of Hoechst-
detectors are set so that the bulk identifiable population
is centered when Hoechst-Blue is displayed on the Y axis,
and Hoechst-red is displayed on the X axis. Red blood
cells show up in the far lower left corner as cells which
indicate little or no Hoechst fluorescence. With the W
laser in the first position, the red blood cells can be
thresholded out. On an Enterprise laser, the W beam is in
the second position, so this is not possible. Dead cells
fluoresce with propidium iodide, and show up on the far
~ CA 02223492 1997-12-04
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. . ,
; .
-23-
right of the profile, as very positive on the red axis. A
live gate is drawn to include only live nucleated cells on
-the basis of the above parameters. In order to identify
the region cont~ining the stem cells, a sufficient number
-5 of data points (i.e., events) must be collected during flow
cytometry analysis (ioo~ooo events, where 10,000 events is
st~Ard~l. This allows the small side-population (SP) to
be readi]Ly identified, as shown boxed in Figure lA. When
this regiLon is purified by fluorescence activated cell
sorting l,FACs) and when used in transplantation assays,
describecl below, this region contains all of the stem cell
activity in C57Bl/6 mice. The frequency of cells in this
region is close to 0.1~.
.. . . . . .
Cell Surface Characterization of the Hoechst "Side
Po~ulatic~n"
Antibody staining was performed as follows: Hoechst-
stained bone marrow was suspended in Hanks Balance~ Salt
Solution (HBSS) containing 2% fetal calf serum, lm~ HEPES,
penicillin, and streptomycin (HBSS+) at lo8 cells per ml.
The antibodies that make up the lineage cocktail w~ere added
at 1/50 t:o 1/100 dilutions (after being titered for this
cell conc:entration). The cocktail is comprised of the
following: CD4 (GK1.5, Becton Dickenson); CD8 (53~6.7,
Becton Dickenson); CD5 (53-7.3, Pharmingen); B220 (RA3-6B2,
Caltag); Mac-l (Ml/70.15, Caltag); Gr-1 (RB6-8C5,
Pharmingen). The mixture was incubated on ice for 10
minutes, then the bone marrow was washed once in excess
HBSS+ ancl the cells were pelleted through a serum cushion.
All washes were performed in this manner. The cells were
resuspencled in media containing Goat anti-rat antibody
conjugated to phycoerythrin (mouse-serum adsorbed, Caltag),
and incubated for 10 minutes on ice. After washing, the
cells were resuspended in 1/3 volume rat serum (Cappel) and
CA 02223492 l997-l2-04
W O 96~9489 PCTAJS96/09415
2/3 HBSS+. After 10 minutes on ice, biotinylated Sca-l
antibody (E13 161-7) was added for 10 minutes on ice.
After washing, the cells were stained with avidin-FITC
(Becton Dickenson) for 10 minutes on ice. Alternatively,
Goat-anti-rate-FITC may be used to detect the lineage
antibodies, and streptavadin-PE for Sca-l.
After the final wash, the bone marrow cells were
resuspended in HBSS+ containing 2~g/ml propidium iodide
(PI). The addition of PI did not affect the Hoechst
staining profile as shown in Fig. lA, but allowed exclusion
of dead cells as described above. In sorting experiments,
the bone marrow was sometimes magnetically pre-enriched for
Sca-1 positive cells using the MACS (Miltenyi Biotec) and
streptavidin microbeads. This resulted in a 5 to 10 fold
enrichment, and did not affect enrichment data. Cells were
sorted into glass tubes containing 100% fetal calf serum.
An aliquot was removed and reanalyzed to establish high
purity, and cells were washed and counted prior to dilution
for bone marrow transplantation.
The Sca/lin profile on whole bone marrow is shown in
Figure lB with the region shown by us and others to contain
all of the stem cell activity in the mouse indicated. This
region contains between 1-5% of the cells in the bone
marrow after live gating out the red blood cells and dead
cells as described above. This number varies depending on
which secondary conjugates are used to detect Sca-l or the
lineage cocktail.
If the region indicated in Fig. lB is used as a live
gate (in conjunction with the gate to exclude red and dead
cells), the Hoechst profile is heterogeneous as shown in
Fig. lC. 5-10% of the cells fall into the side population.
If the region indicated in Figure lA is used as the SOLE
LIVE GATE on the whole population of murine bone marrow
cells, the sca/lin profile of those cells is shown in
Figure lD. In the example shown, approximately 75% of the
. ~ CA 02223492 1997-12-04
W O 96J39489 = PCTJUS96JD94
~. ,
, ~. ~ . ...
-25-
cells faLl into the Sca-l~Ve lineage~-~e region shown to
contain aLll of the stem cells. Reducing the size of the
~ side population box to avoid contamination from the bulk
population increases the apparent cell surface bLomogeneity
of the SP cells up to about 95%.
Thi-~ procedure to characterize the cell surface
expression of the Sca-l and lineage antigens can be
repeated for any marker alone or in combination. The
following characterizations have thus been made for the
human SP cells: glycophorin A~, CD38l~/n~, CD13~, CD15~,
CD19~, CD20n~, and CD34~. The following characterizations
have been made for the murine SP cells: c-kit~, 5ca-1~,
Gr-l~/~, Mac-ln~, CD4 n~/~ CD8~, B220n~, CD5n~, CD43~,
CD45~, M169~, AA4D~/~, class IMHC~, class II MHC~,
Rhodamine 123~ and Wheat germ aLgglutinin (WGA)~
~xam~le .!: Lonq Term Bone Marrow Trans~lants Usinlq
Hoechst-~urified stem cells
Com~etitive RePopulation Assay Usinq Hoechst-~urified stem
cells
To examine the functional properties of the cells
purified in Example 1, long term bone marrow
transplantation experiments were performed. For the
competitive repopulation assay congenic C57Bl/6 mouse
strains which differ at the Ly-5 locus were used (Scheid,
M.P. and Triglia, D., Immunogenetics, 9:423-433 (1979)).
The Ly-5 antigen is found on the surface of all nucleated
peripheral blood cells and the two allelic variants (i.e.,
Ly-5.1 and Ly-5.2) are readily distinguished by specific
monoclona,l antibodies. Peripheral blood was collected from
transplant recipients at multiple time points and assayed
by flow cytometry for the proportion of the two Ly--5
alleles present in each blood lineage.
.. . . ..
.
.
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W O 96/39489 PCT~US96/09415
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Typically, bone marrow was extracted from femurs and
tibias of 10 male C57B1/6-Ly-5.1 mice (National Cancer
Institute) 6-10 weeks in age and purified as described in
Example 1. Stem cells were purified on the basis of the
combination of the Hoechst side population and Sca-l+lin~
fluorescence, as described in Example 1. This population
was selected as follows: first, a live gate was defined
using Hoechst red and blue axis to exclude dead cells and
debris. After collecting 105 events within this live gate,
the SP population is able to be clearly defined. A new
live gate is established on this population, as shown boxed
in Figure lA, and the fluorescence of Sca-1 and lineage
markers on SP cells was displayed. This allows a clear
definition of Sca-1+ Lin~ cells, as the bulk of the SP
cells stand out immediately as falling into the region
defined in Figure lB. A sorting gate is then defined as
cells which fall into both the region boxed in Figure lA
and in Figure lB. Cells are purified by FACs and when they
are reanalyzed, the purity is greater than 98%.
These purified cells were counted and mixed with
unfractionated bone marrow cells (i.e., competitor cells)
obtained from two male C57B1/6-Ly-5.2 mice (Jackson
Laboratory), 6-10 weeks in age, and the mixtures were
introduced into female C57B1/6-Ly-5.2 recipient mice (i.e.,
recipients) (Jackson Laboratory). The recipients were 6 to
12 weeks in age, and maintained on acidified water. The
recipients were irradiated with 1100 rads given in two
doses (620 rads and 480 rads) at least 2 hours apart.
Transplanted cells were given intravenously by retro-
orbital injection under methoxyflurane (Pittman Moore)anesthetic. Peripheral blood was taken by retro-orbital
puncture under methoxyflurane anesthesia 4 months post-
transplant. All animal care was in accordance with
institutional guidelines. Red blood cells were removed and
~ CA 02223492 1997-12-04
W ~ 96/39489 =~ PCTrUS96109415
,' ' . '~ ' '' - " '
~ 27-
the nucleated peripheral blood cells were stained with
biotinylated anti-Ly-5.1 antibody which was detected with
- streptavidin-PE (Molecular Probes). Sometimes, the blood
samples ~Jere subdivided and co-stained with directly
- 5 conjugat~d lineage specific antibodies against Thy-1, B220,
Gr-l, ancl/or Mac-1 (Pharmingen). These blood samples were
analyzed on the Facstar-plus, or a Facscan (Becton
Dickensorl). The anti-Ly-5.2 and anti-Ly-5.1 hybri~omas
used were 104.2.1 and A20.1.7 respectively (gifts from D.
Pardoll). Other anti-Ly-5.2 and anti-Ly-5.1 hybridomers
are avai].able for use (Spangrude, G.J., et al., Sc.ience,
241:58-62 (1988)).
The results are shown in Table 1. The first column
displays the number of Hoechst-purified stem cells (HSC).
The second column displays the number of competitor cells
introduced (see below). N represents the number o:E
transplarlt recipients at the time of analysis (usually the
same as t:he number initially transplanted) in each group.
The mean is the mean number of HSC-derived (Ly-5.1-~)
nucleated peripheral blood cells present in recipients 4
months post-transplant, and SD is the standard dev:iation.
Enrichment is calculated as the mean percentage
contribut.ion Ly-5.1+ cells in the peripheral blood per
purified cell introduced, divided by the mean percentage
contribut.ion of Ly-5.2+ cells in the peripheral blood per
unfractionated Ly-5.2 cell introduced. All numbers are
rounded t.o two significant digits.
As s.hown in Table 1, the peripheral blood was analyzed
4 months post-transplant, ensuring that most of the cells
present w-ere derived from HSC. Furthermore, the
contribution of the SP cells to the total stem cell
activity was maintained at the same level for at least 12
months post transplant. In addition, the SP cells have
been shown to contribute to all major blood lineages.
Interestingly, as seen in Table 1, lower does of purified
. ~ :
CA 02223492 1997-12-04
W O 96/39489 PCTtUS96tO9415
-28-
SP cells routinely appear to give rise to apparently higher
enrichments, suggesting that there is some complex
regulation when large numbers of purified stem cells are
introduced into recipients.
CA 02223492 1997-12-04
WO 96/39489 P~TIUS96J0941
. . .
-29-
J~
0 0 0 0 0 0 o
~ ~ c~ o o 1~ O
V ~1
Ll
C ~ .
_, 00
cn
~C
a a~ D c~
._ ~
~3
o~
C)
3:
R
r
o
0 ~ o o o o o o
o o o o o o
O N t~l C~l ~r ~ ~r
a~ ~ X
.,~ o
O
O O O O o o
O ~q ~ O
0
_I
.
-
CA 02223492 1997-12-04
W O 96/39489 PCT~US96/09415
-30-
Com~etitive repo~ulation assav usinq various reqions of the
hemato~oietic stem cell poPulations in Fiqure lE
Each of the three regions in Figure lE was purified as
described in Example 1 and tested for stem cell activity
using the competitive repopulation assay described above.
Bone marrow from C57B1/9-Ly-5.1 mice was magnetically
enriched for Sca-l antibody staining cells to afford
approximately 8-fold enrichment for stem cell activity.
Sca-1 is known to mark all of the stem cells in this strain
of mice.
The results are shown in Table 2. The first column
indicates the region of cells tested. The second column
displays the number of purified cells transplanted. The
third column displays the number of competitor cells
introduced. N represents the number of transplant
recipients at the time of analysis (usually the same as the
number initially transplanted) in each group. The mean is
the mean number of nucleated peripheral blood cells derived
from the fractionated bone marrow present in recipients 4
months post-transplant, and SD is the standard deviation.
Enrichment is calculated as the mean % Ly-5.1+ cells in the
peripheral blood per purified cell introduced, divided by
the mean percentage contribution of Ly-5.2+ cells per
unfractionated Ly-5.2 cell introduced. All numbers are
rounded to two significant digits.
CA 02223492 l997-l2-04
W O 96/39489 PCTAUS96/094~5
-31-
J~
~ O CO O 0~ N
cn ~o N
~ V .C ~D ~iD C/~
U
a, ~
W
U~
u~ un o~
o
O
.,1 ~
P.
~ U~
E~
C
u ~ un
~n
~ X
C~ o o o o
C N ~ O O O O O
c . ,, C~ o o ~n In
O
O
X
tl) I~') N
11 ~ . O
O 0
_
~)
o
C~
04
O U~ 04
~ ~rl U'~
N ~ O.l 04 04
U u~ U~ U~
_ o
R
E~
CA 02223492 1997-12-04
W O 96/39489 PCT~US96/09415
-32-
Radio~rotective ability of ~urified stem cells
Having established average enrichments of HSC activity
of approximately 1000 fold with the SP cells in the
competitive repopulation assays, the ability of the SP
cells to protect recipients from lethal irradiation was
investigated.
Purified HSC (male C57B1/6-Ly-5.1 derived, Hoechst SP,
Sca-1+, lin~) were introduced into female C57Bl/6-Ly-5.2
recipients as described above for the competitive
repopulation assay. Results from three independent
experiments are shown in Table 3. The number of purified
HSCs introduced into the recipient mice are shown in the
first column. The number of animals transplanted in each
group is shown in the second column. The percentage of
animals surviving at least 4 months post-transplantation is
shown in the third column. The mean percent Ly-5.1
nucleated peripheral blood cells in the transplant
recipients 4 months post-transplant is shown in column
four. The st~n~Ard deviation (SD) is shown in column five.
All numbers are rounded to two significant digits.
As shown by data from three separate experiments in
Table 3, approximately 150 purified cells radioprotected
50% of recipients. The mean contribution of the SP cells
to hematopoiesis in the peripheral blood of survivors was
at least 81%. Although others have observed a higher level
of radioprotective activity from their purified
populations, the mean contribution of the transplanted stem
cells to hematopoiesis in their survivors was considerably
lower (Spangrude, G.J., et al., Science, 241: 58-62 (1988);
Fleming, W.H., et al., ~. Cell Biol., 122: 897-902 (1993);
Li, C.L., Johnson, G.R., Exp. Hematol ., 20:1309-15 (1992);
Spangrude, G.J. and Scollay, R., Exp. ~ematol ., 18: 920-926
(1990)). This may reflect qualitative differences in the
purified populations, or possibly differences in animal
- ~ CA 02223492 1997-12-04 ..
W0 96~39489 ~ PC'r/US96/094f~;
, -
~ 3~
.
husbandry practices which affect survival after lethal
irradiation. In addition, the 12-day spleen colony
formation (CFU-s) of this population was measured to be
approximately 1 CFU-s(l2) per 35 purified cells injected.
This frequency correlates well with the CFU-s( 12) ~orming
frequency of the Rhodamine-123~ sub-population of Sca-1+
lin~ Thy-l.l~ cells (Spangrude, G.J. and Johnson, G.R.,
Proc. Nal'l . Acad. Sc~ . U.S.A., 87:74-3-7 ~1990~).
c -- - -
. . .
.
.
-,
~; .
. = ~
.
CA 02223492 1997-12-04
W O 96/39489 PCTrUS96/0941
-34-
_I O ~D I I t~
t~
o t,~ ~ tJ~ ,1 0 t~ t~ O U~ ~ t~
~ ~ t~ 0 t~ ~ t~ tn tn tn
-
CJ
tn d ~ ~ I~ ~ O ~r o ~r O o o ~) o t~
t,~ tX~ t' t~
,a _,
s~ tn
d~
o
o o o o o o tn tn o ~ o o o tn
t.~ ~ t,~ ,I t,~ t.~ _~ t~ t~l t,~
.~
-
-
o o o ou~ o o o o o o O O
t ,~ooo t~In oo o~oo
P ,~ t~ I t~ _/ t~ ~ o
a
t
E~
- -
~ -CA 02223492 1997-12-04
- ,,
WO 96~39489 PCT~US96/094~;
-35-
Example 3: Inhibition of Multi~le Druq Resistant ~rotein
With Vera~amil
Sinl-e it is known that several vital dyes are actively
pumped out of the cells by the multidrug resistance protein
(mdr, or p-glycoprotein), the possibility that the
exceptio~ally low Hoechst fluorescence was due to higher
mdr acti~ity in murine hematopoietic stem cells was
investigated (Chaudhary, P.M. and Roninson, L.B., Cell ,
66:85-94 (1991)).
Who:Le murine bone marrow was stained for 90 minutes in
5~g/ml Hoechst 33342 as described in Example 1 with 50 ~M
verapami:L or without verapamil (Sigma). After staining,
samples were kept on ice until flow cytometry analysis as
describecl in Example 1.
The results, which are shown in Figures 2A (without
verapami]L) and 2B (with verapamil), demonstrate tha~
purified stem cell population cannot be isolated in the
presence of verapamil using the method of the present
invention. Therefore, it is reasonable to expect that the
stem cells are uniquely low in Hoechst fluorescenc~e due to
mdr or an mdr-like mediated efflux of the dye. This very
high dye efflux activity may be due to a higher level of p-
glycoprot:ein on the surface of HSC, a higher level of
activity of the p-glycoprotein present, or an mdr-Like
activity not yet identified. Antibody studies on the
expression of the product of the MDR1 gene in humal~
hematopoietic cells suggest that p-glycoprotein is in fact
expressed~ quite widely, and that up to 65% of bone marrow
cells may express mdr (Chaudhary, P.M. and Roninson, L.B.,
30 Cell, 66:85-94 (1991); Drach, D., et al., Blood, 8~:2729-34
(1991)). In light of this, a purification strategy based
on functional properties, such as the one described herein,
will likely be more powerful than a scheme based on the
level of mdr cell surface expression (e.g., antibody-
3 5 based).
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Exam~le 4: AnalYsis of the Cell Cycle Status of Purified
Hemato~oietic Stem Cells
Initial purification of the HSC was as described in
Example 1. After sorting to 98% purity, several thousand
sorted stem cells were incubated for 60 minutes at 37~C in
10 ml DMEM containing 2% fetal calf serum, lmM HEPES,
Penicillin, Streptomycin, 10 ~g per ml Hoechst 33342, and
50 ~M verapamil. Flow cytometry analysis was as described
in Example 1. Propidium iodide stain: Stem cells purified
as described above were pelleted by centrifugation and
resuspended in 0.1% NaCitrate, 50 ~g/ml propidium iodide.
After incubation on ice for 10 minutes, the cells were
analyzed by standard flow cytometry procedures using 488 nm
excitation. The results are shown in Fig. 3A and Fig. 3B.
An aliquot of the SP cells was removed and
transplanted as the "lx sorted" group in column 1. The
rest of the sorted cells were incubated for 60 minutes in
DMEM as described in Figure 1, containing 10 ~g/ml Hoechst
33342 and 50 ~M Verapamil. These were resuspended in HBSS
including 2 ~g/ml of propidium iodide, and sorted on the
flow cytometer as described in Figure 1. Several thousand
cells were first allowed to pass through the laser without
subfractionation. These were transplanted as the "2x
sorted" group. Large differences in the reconstitution
potential between the lx sorted and 2x sorted groups could
have indicated damage of the HSC by the sorting conditions.
The remaining cells were sorted into either 2n (Go~GI) or
>2n (S-G2M) groups, and transplanted as indicated.
When cell-cycle analysis was performed in this way,
the number of cells in S-G2M (i.e., cycling HSCs) was found
to range between 1 and 3% of the purified cells (Fig. 3A).
This figure correlates well with the number shown to be in
S-G2M by propidium iodide staining of the purified cells
(Fig. 3B). This is much lower than the fraction of cells
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-3~-
in S-G2M in whole bone marrow (20%), and in other
populations enriched for stem cells (Fleming, W.H., et al.,
J. Cell ~iol ., 122:897-902 (1993)). This low number of
cells in cycle supports the widely held view that ~SC from
normal bone marrow are largely quiescent.
With a view to using cycling stem cells as targets for
retrovirzll-mediated gene transfer, the relative engraftment
potentia] of stem cells that were in Go~GI (i.e., quiescent
HSCs) versus S-G2M was compared. These subsets of the SP
population were purified by flow cytometry using the
verapamil-blocking strategy for cell cycle analysis
describecl in Example 1, and tested in the competitive
repopulation assay described in Example 2. Table 4 shows
the results from the experiment.
One experiment is shown with the analysis per~Eormed at
both 2 months and 11 months post-transplant. The number of
purified or sub-fractionated HSC transplanted per mouse is
shown in the second column (HSC). The number of
unfractionated Ly-5.2 competitor bone marrow cells co-
transplan,ted is indicated in the third column, and the
number of mice at the time of analysis (n) in the fourth
column. The mean shown is the mean percentage of Ly-5.1+
nucleated peripheral blood cells at the time post-
transplant indicated, and SD is the standard deviation.
The enrichment is calculated as in Table 1. All numbers
are rounded to two significant digits.
These data demonstrate several points. First]y, the
contribution of the Go~GI HSC to the peripheral blood is
almost identical to that of the S-G2M HSC, and both of
these populations are virtually identical in activity to
HSC which have not been sub-fractionated (lx and 2xx sorted
HSC). Secondly, a passage of HSCs through the flow
cytometer twice (2x sorted; exciting the fluorescence of a
DNA-binding dye with a W laser) does not seem to affect
,:
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-38-
the capacity of the HSC to repopulate the bone marrow.
Thirdly, the percentage of peripheral blood cells derived
from the sorted cells is almost identical at 2 months and
11 months, reflecting the very long term stem cell activity
of all of these populations.
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WO 96139489 PCT/U~;96/0941
--39--
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--~0--
~ple 5: Characterization of Hoechst 33342 Fluorescence
on Whole Human Bone Marrow and Human Cord Blood
Cell surface characterization of human SP cells
Hematopoietic stem cells from whole human bone marrow
and human umbilical cord blood were identified as described
in Example 1, except as described below. Human SP cells
were characterized following the procedure for cell surface
characterization of the mouse SP cells described in Example
1. In some samples, there are some CD34-positive cells at
the TOP of the SP region, near the bulk population of
cells. But the majority of cells in the SP region in
several samples of human marrow that we have ~A~; ned are
characterized as follows: Glycophorin An~, CD38~, CD13n~,
CD15n~, CD19n~, CD20n~ and CD34~. The human cells run at a
lower frequency, closer to 0.03-0.05% in the bone marrow.
Typically, this bone marrow has been depleted of red blood
cells and platelets, by a ficoll density centrifugation.
The results are shown in Figures 4A and 4B.
E~am~le 6: Characterization of Hoechst 33342 Fluorescence
Qn Porcine Bone Marrow and MonkeY Bone Marrow
Hematopoietic stem cells from porcine bone marrow and
monkey bone marrow were identified as described in
Example 1, except as indicated below. Porcine bone marrow
and Rhesus macaque monkey bone marrow were characterized
following the procedure for cell surface characterization
of the mouse SP cells described in Example 1. In addition,
fresh bone marrow was depleted of red blood cells in the
case of Rhesus bone marrow by a ficoll density gradient.
Nucleated cells were counted and resuspended at 106
nucleated cells per ml in pre-warmed DMEM/10% FCS, lmM
HEPES, and 50 units per ml penicillin and 50 ~g per ml
streptomycin. Hoechst 33342 dye was added to a
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concentration of 5 ~g/ml, and the cells were incubated at
37~C for 120 minutes. Bone marrow samples were run on the
flow cytometer using the set-up described for the murine
bone marrow.
The results are shown in Figures 5A and 5B. The
arrows indicate the presence of cells in the same region
murine s1:em cell activity is observed (see Figure lA). The
region represents 0.05% to 0.1% of the nucleated cell
population in both of these animal models. The phenotype
of the ~lesus monkey SP cells are as follows: CD34~,
CD38~/D~, CD4n~, CD8n~, Glycophorin A negative, CD6ln~, and
CD66n~.
~ m~le ,': Rhesus Lonq Term Culture Initiating Cell
(LTCIC) I,imitinq Dilution Experiments
The LTCIC assay is considered by many in the field to
represent: the best in vitro assay for HSCs. As described
below, this assay was performed on hematopoietic stem cells
from Rhesus monkeys. Hematopoietic stem cells from Rhesus
monkeys were identified as described in Example l. The
LTCIC assays were performed as described in Sutherland,
H.J., et al., Proc. Natl. Acad. Sci., 87:3584-3588 (l990).
The results are shown in Table 5.
Table 5. Rhesus LTCIC Limiting Dilution Experimen~s.
UnfractionatedUnfract./H0- CD34~'/CD38n~ 8P+/CD34
stained
l/4 x 103 l/5 x 103 l/55 l/6
1/12 ~ 103 l/15 x 103 l/53 l/8
1/12 ~' 103 1/8 X 103 1/51 1/7
-
Numbers given represent number of LTCIC forming units
per cell plated in a limiting dilution-type assay. 80ne
marrow from three separate monkeys is shown. As shown in
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Table 5, the hematopoietic stem cell activity on a per cell
basis of the SP cells that are CD34 negative (column 4) is
significantly higher than the CD34P~'/CD34~~ cells
(column 3), which are considered in the art to define stem
cells, from the same animal. The controls are
unfractionated (column 1) and unfractionated/Hoechst-
stained (column 2) marrow. The unfractionated and CD34P~'hh'numbers of LTCIC are right in the range of LTCIC activity
found in these populations in the literature. (Sutherland,
H.J., et al., Proc. Natl. Acad. sci., 87:3584-3588 (199O)).
Thus, the data indicates that in the monkey, SP cells,
which are CD34~~, are highly enriched for LTICs.
EOUIVALENTS
Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention
described specifically herein. Such equivalents are
intended to be encompassed in the scope of the following
claims.
