Note: Descriptions are shown in the official language in which they were submitted.
208025~
Title: PRIMITIVE HEMATOPOIETIC ST~M CELL PREPARATIONS
FIELD OF THE INVENTION
The invention relates to the isolation and use of
primitive hematopoietic stem cells from leuke~ic patients
and from blood of normal individuals; quantitative assays
for same; and, methods for testing for substances which
affect hematopoiesis of the hematopoietic stem cells.
BACgGROVND OF THE INVENTION
The regenerative capacity and life-long maintenance of the
hematopoietic system is dependent on a primitive
subpopulation of stem cells with extensive self-renewal,
proliferative and differentiation potential. Totipotent
hematopoietic stem cells with the capacity to repopulate
lymphoid and myeloid tissue in myeloablated recipients
have been documented (Abramson, S. Miller, R.G. h Philips,
R.A., J. Exp. Med., Vol. 145, pp. 1567-1579, 1977; Mintz,
B. Anthony, K. & Litwin, S. Proc. Natl. Acad. Sci USA,
Vol. 81, pp. 7835-7839, 1984; Turhan et al., N. Engl. J.
Med., Vol. 320, pp. 1655-1661, 1989; Keller et al.,
Nature, Vol. 318, pp. 149-154, 1985; Dick et al., Cell,
Vol. 42, pp. 71-79, 1985; Lemischka et al., Cell, Vol. 45,
pp. 917-927, 1986; Snodgrass, R. & Keller, G., EMBO J.,
Vol. 6, pp. 3955-3960, 1987. Capel et al., Proc. Natl.
Acad. Sci. USA, Vol. 86, pp. 4564-4568, 1989; Capel et
al., Blood, Vol. 75, pp. 2267-2270, 1990; Van Zant, et
al., Blood, Vol. 77, pp. 756-763, 1991).
- Evidence points to a hierarchy of stem cells with
differing potentials for sustaining hematopoiesis when
transplanted in vivo. Cells with long term hematopoietic
reconstituting ability can be distinguished by a number of
physical and biological properties from cells that only
generate mature progeny in short-term in vivo or in vitro
clonogenic assays (Hodgson, G.S. & Bradley, T.R., Nature,
Vol. 281, pp. 381-382; Visser et al., J. Exp. Med., Vol.
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59, pp. 1576-1590, 1984; Spangrude et al., Science, Vol.
241, pp. 58-62, 1988; Szilvassy et al., Blood, Vol. 74,
pp. 930-939, 1989; Ploemacher, R.E. & Brons, R.H.C., Exp.
Hematol., Vol. 17, pp. 263-266, 1989).
There have been substantial efforts to develop methods to
detect, isolate and characterize the most primitive
hematopoietic stem cells. Such cell populations are
expected to be useful in bone marrow transplants for the
treatment of a variety of conditions such as AIDS,
leukemia and certain anemias, and in gene therapy. The
cell populations may also be used to identify growth
factors, to screen growth factors and in assays to study
the development of hematopoietic cells.
Primitive hematopoietic stem cells capable of initiating
long term culture have been identified. The number of
clonogenic cells present after 5 to 8 weeks in long term
cultures initiated with normal hematopoietic cells allows
the detection of a very primitive class of clonogenic cell
precursors that exhibit properties characteristic of cells
with long-term in vivo reconstituting potential
(Sutherland et al., Blood, Vol. 74, p. 1563, 1986 -
Udomsakdi et al., Exp. Hematol., Vol. 19, p. 338, 1991.)
These normal human "long-term culture-initiating cells"
(LTC-IC~ can be quantitated by limiting dilution analysis,
which then allows the proliferative potential of
individual LTC-IC to also be determined (Sutherland et
al., Proc. Natl. Acad. Sci., Vol. 87, p. 3584, 1990).
Normal marrow LTC-IC are well maintained in LTC
estab.lished from a single input inoculum (Eaves et al.,
Effects of Therapy on Biology and Kinetics of Residual
Tumor, Part A: Pre-Clinical Aspects, p. 223, 1990 - Eaves
et al., Ann. N.Y. Acad. Sci. Vol. 628, p. 298, 1991) and
similar kinetics are seen when highly purified LTC~IC from
normal marrow are seeded onto preestablished feeders
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(Sutherland et al., Proc. Natl. Acad. Sci., Vol. 87, p.
3584, 1990).
Abnormalities of the primitive hematopoietic stem cells
- may give rise to a variety of conditions, including
leukemia, a family of disorders characterized by the
progressive proliferation of abnormal leukocytes. Chronic
- myeloid leukemia (CML) is a multi-lineage clonal
hematopoietic malignancy characterized by excessive
production of granulocytes and the presence in the
leukemic cells of a consistent rearrangement of the BCR
and ABL genes, typically manifested in metaphase
preparations as the Philadelphia chromosome (Ph1) (Goldman,
Balliere's Clinical Haematology, Vol. 1, 1987). The
initial cell transformed and hence the origin of the
leukemic clone is believed to be a totipotent
hematopoietic cell with lymphoid as well as myeloid
differentiation potential. (Fialkow et al., Proc. Natl.
Acad. Sci., Vol. 58. p. 1468, 1967). The most primitive
hematopoietic cells are difficult to study because they
make up such a small proportion of all the nucleated cells
in the blood and marrow. Nevertheless, effects of BCR-ABL
eY.pression in their behaviour are of key interest because
it is th~se cells that are believed to be responsible for
the initial amplification of the leukemic clones in
patients with CML.
CML patients with elevated white blood cell (WBC) counts
show dramatic increases in the number of Ph1-positive
clonogenic progenitors in their circulation (Eaves et al.,
Bailliere's Clinical Haematology Vol. 4, pg. 931, 1990 -
Dowding et al. Int. J. Cell. Cloning Vol. 4, p. 331,
1986). Continued production of Ph1-positive clonogenic
cells for many weeks can occur at a high level when
peripheral blood cells from such patients are cultured on
irradiated marrow cell adherent layers established from
normal individuals (Eaves et al., Proc. Natl. Acad. Sci.,
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Vol. 83, p. 5306, l986).
Allogenic bone marrow transplantation is a useful therapy
for some leukemias, including CML, although the procedure
is limited by the availability of compatible donors.
Autologous bone marrow transplantation is a less useful
treatment for leukemia in general, and CML in particular,
due to the difficulties of o~taining a bone marrow
preparation which is devoid of primitive hematopoietic
stem cells of leukemic origin and which may proliferate in
the donor causing relapse. Studies of primitive leukemic
hematopoietic cells have been limited by the lack of a
suitable assay for their identification, and of methods
for their selective isolation or ablation. In particular,
the biological consequences of BCR-ABL kinase expression
in very primitive human hematopoietic cells has been
difficult to investigate because methods for their
selective isolation have not been avAilable.
SUMMARY OF THE INVENTION
The present inventors have isolated the most primitive
human hematopoietic stem cells yet defined from patients
with chronic myeloid leukemia. It was surprisingly found
that a highly enriched population of these cells could he
obtained from the blood of CML patients with increased
white blood cell counts. The primitive leukemic
hematopoietic stem cells were found to be capable of
generating clonogenic cells, on average 3-4 clonogenic
cells, after at least 5 weeks of culture on competent
feeder cells and accordingly are alternatively referred to
herein as CML long term culture initiating cells (CML LTC-
IC). The cells were also found to be relatively 4-hydroxy-
cyclophosphamide sensitive, HLA-DR positive, rhodamine-
bright and larger in size (higher FLS) than normal
peripheral blood LTC-IC. The present inventors were also
able to obtain a cell preparation having a purity of
circulating CML LTC-IC of approximately 10~, which is 5 to
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6 fold higher than the purest populations of normal LTC-IC
thus far isolated from normal blood or marrow samples
(Lansdorp PN et al, J Exp Med 172:363, 1990; Udomsakdi C
et al, Exp Hematol 19:338,1991; Sutherland HJ et al PNAS
USA 87: 3584, 1990).
The present inventors also have surprisingly found that
CML LTC-IC cells can be quantitated by measurement of the
number of clonogenic cells produced after at least 5 weeks
in culture on competent fibroblasts. A linear relationship
was found for the number of clonogenic cells present 5
weeks after seeding light density peripheral cells from
CNL patients with high numbers of circulating Ph1 positive
clonogenic cells onto irradiated normal marrow adherent
layers and the number of peripheral blood cells initially
added. Neasurement of the frequency of CML LTC-IC by
limiting dilution analysis allowed derivation of their
average clonogenic cell output at the 5 week time point.
The present inventors further surprisingly found that CML
LTC-IC are selectively disadvantaged in long term culture
compared to normal LTC-IC.
Furthermore, the present inventors have found primitive
hematopoietic stem cells i.e. LTC-IC, in normal blood.
- They have significantly shown that the number of
clonogenic cells present in long term cultures of T cell-
depleted fractions of normal blood after 5 weeks, is
linearly related to the input number of peripheral blood
cells over a wide range of cell concentrations, thereby
permitting the quantitation of circulating LTC-IC by
limiting dilution analysis. Using this approach, the
concentration of LTC-IC in the circulation of normal
adults was found to be 2.9 + 0.5 per ml. This is about 75-
fold lower than the concentration of circulating
clonogenic cells and represents a frequency of LTC-IC
relative to all nucleated cells that is about 100 fold
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lower than that measured in normal marrow aspirate
samples. Characterization studies revealed most
circulating LTC-IC to be small (low forward light scatter
and side scatter), CD34+, Rh-123dUll, HLA-DR- and 4-
S hydroperoxycyclophosphamide-resistant cells with
differentiative and proliferative potentialities similar
to LTC-IC in normal marrow. Isolation of the light-
density, T-cell-depleted, CD34+, and either HLA-DR low or
Rh-123dUll fraction of normal blood yielded a highly
enriched population of cells that were 0.5 - 1% LTC-IC, a
purity which is comparable to the most enriched
populations of human marrow LTC-IC reported to date.
Therefore, the present invention relates to a cell
preparation comprising primitive hematopoietic stem cells
obtained from leukemic patients which cells are
Philadelphia chromosome positive and produce detectable
clonogenic progenitors in long term culture. In an
embodiment of the invention, the cell preparation is
obtained from the blood of CML patients with increased
white blood cell counts and the primitive hematopoietic
stem cells in the preparation are CD34~and HLA-DR+, FSChi9h,
SCC~W, and Rh-123~Ve. Preferably the cell preparation has a
purity of circulating primitive leukemic hematopoietic
stem cells of approximately 10%.
The invention further provides a cell preparation
comprising primitive hematopoietic stem cells obtained
from the blood of normal individuals which cells produce
detectable clonogenic progenitors in long term culture. In
an embodiment of the invention, the primitive
hematopoietic stem cells in the preparation are CD34'and
HLA-DR-, ~sc.Ow SCCLW, and Rh-123dU~ and 4-
hydroperoxycyclophosphamide-resistantwithdifferentiative
and proliferative potentialities similar to LTC-IC in
normal marrow. Preferably the cell preparation has a
purity of circulating primitive hematopoietic stem cells
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of approximatel~ 0.5 - 1%.
The invention also provides a method for preparing a cell
preparation comprising primitive hematopoietic ~tem cells
obtained from leukemic patients which cells are
Philadelphia chromosome positive and produce detectable
clonogenic progenitors in long term culture comprising
obtaining a sample from a leukemic patient, preferably a
blood sample from a leukemic patient having an elevated
white blood cell count, most preferably having a white
blood cell count greater than 20 x 109 white blood cells
per litre of blood; and isolating a cell preparation from
the cell sample which contains cells which are
Philadelphia chromosome positive and produces detectable
clonogenic progenitors in long term culture. Preferably,
the cell preparation obtained substantially comprises
primitive leukemic hematopoietic stem cells which are
CD34' HLA-DR+, FSChi9h, SCCLW, and Rh-123'Ve. Preferably, a
cell preparation having a purity of circulating primitive
leukemic hematopoietic stem cells of approximately 10% is
obtained.
The invention further provides a method for preparing a
cell preparation comprising primitive hematopoietic stem
cells obtained from the blood of normal individuals which
cells produce detectable clonogenic progenitors in long
term culture comprising obtaining a blood sample from a
normal individual, and isolating a cell preparation from
the cell sample which contains cells which produce
detectable clonogenic progenitors in long term culture.
Preferably, the cell preparation obtained substantially
comprises primitive hematopoietic stem cells which are
CD34~ and HLA-DR-, FSC~Wl SCClW, Rh-123dUL~ and 4-
hydroperoxycyclophosphamide-resistantwithdifferentiative
and proliferative potentialities similar to LTC-IC in
normal marrow. Preferably, a cell preparation having a
purity of circulating primitive hematopoietic stem cells
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of approximately 0.5 to 1% is obtained.
The invention also relates to a method for quantitating
primitive leukemic hematopoietic stem cells in patients
with leukemia comprising obtaining a sample which contains
primitive leukemic hematopoietic stem cells from a
leukemic patient, preferably a blood sample from a
leukemic patient having an elevated white blood cell
count, preferably greater than 20 ~ 10~ white blood cells
per litre of blood; optionally enriching primitive
leukemic hematopoietic stem cells in the sample;
coculturing the sample with a feeder cell layer for at
least five weeks under conditions which permit the
production of clonogenic progenitor cells; harvesting
nonadherent cells and adherent cells; subjecting the
harvested cells to secondary assay culture under suitable
conditions to express clonogenic progenitor cells;
detecting and quantitating the number of clonogenic
progenitor cells; and, quantitating the number of
primitive leukemic hematopoietic stem cells in the sample
on the basis of the linear relationship between the number
of clonogenic progenitor cells and the number of primitive
leukemic hematopoietic stem cells initially in the sample.
The advantages of the above described method of the
present invention include its relative simplicity, ease of
standardization, and applicability to quantitation of
primitive hematopoietic cells in primary patient samples.
Accordingly, the method may be used in the diagnosis of
CML and to characterize the disease state of a patient.
Treatments for leukemia may be evaluated by determining
the number and characteristics of primitive hematopoietic
leukemic stem cells in the blood and bone marrow of a
patient at time periods before and after treatment using
the methods of the invention. The method may also provide
a useful system for studying the interactions of normal
and leukemic primitive hematopoietic stem cells in vivo.
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g
The invention further relates to a method for quantitating
primitive hematopoietic stem cells in the blood of normal
individuals comprising obtaining a blood sample which
contains primitive hematopoietic stem cells from a normal
individual, optionally enriching primitive hematopoietic
stem cells in the blood sample; coculturing the sample
with a feeder cell layer for at least five weeks under
conditions which permit the production of clonogenic
progenitor cells; harvesting nonadherent cells and
adherent cells; subjecting the harvested cells to
secondary assay culture under suitable conditions to
express clonogenic progenitor cells; detecting and
quantitating the number of clonogenic progenitor cells;
and, quantitating the number of primitive hematopoietic
stem cells in the blood sample on the basis of the linear
relationship between the number of clonogenic progenitor
cells and the number of primitive hematopoietic stem cells
initially in the sample.
The invention further relates to a method of purging a
mammalian bone marrow sample containing primitive leukemic
hematopoietic stem cells to prepare a bone marrow cell
suspension substantially free of primitive leukemic
hematopoietic stem cells comprising: obtaining a sample of
bone marrow cells from a leukemic patient; substantially
depleting red blood cells in the sample; coculturing the
depleted bone marrow cell sample with a feeder cell layer;
and harvesting and suspending the cultured bone marrow
cells. The bone marrow cell suspension may be further
cultured on a feeder cell layer; and the cultured bone
marrow cells may be harvested and cultured.
The invention still further relates to a system for
testing for a substance that affects hematopoiesis of
primitive hematopoietic stem cells comprising: preparing
a cell suspension that is enriched in primitive
hematopoietic stem cells; coculturing the cell suspension
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with a feeder cell layer for at least 5 weeks in the
presence of a substance which is suspected of affecting
the hematopoiesis of primitive hematopoietic stem cells
and assessing LTC-IC maintenance, clonogenic cell
production, and/or production of nonadherent cells. The
cell suspension may comprise primitive hematopoietic stem
cells obtained from the blood of leukemic patients,
preferably from the blood sample of a leukemic patient
having an elevated white blood cell count, or from the
blood of normal individuals. The substance may be added to
the culture of the cell suspension and feeder cell layer
or the feeder cell layer may be genetically engineered to
express the substance i.e. the feeder cell layer may serve
as an endogenous source of the substance.
DESCRIPTION OF TH~ DRAWINGS
The invention will now be described in relation
to the drawings in which:
Figure 1 is a graph showing the relationship
between the number of CML peripheral blood cells seeded
into long term culture and the number of clonogenic cells
detected after 5 weeks;
Figure 2 is a graph showing limiting dilution
- analysis of CML peripheral blood cells seeded in culture
in decreasing numbers and assayed for clonogenic cells at
5 weeks;
Figure 3 is a graph showing the LTC-IC
concentration in the peripheral blood of CML patients and
normal individuals;
Figure 4 is a graph showing the differential
kinetics of CML (solid symbols) and normal (open symbols)
long term culture initiating cells in vitro;
Figure 5 is a diagram showing the distribution,
according to light scatter characteristics, of total
cells, clonogenic cells and LTC-IC in the light density
fraction of CML blood;
Figure 6 is a diagram showing bivariate contour
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11
plots of a representative sample of normal and CML light
density blood cells in the low SSC window CD34~DR~W,
CD34+DRhi~h CD34+RH-123dUl~andCD34+Rh-123bri9htsubpopulations;
Figure 7 is a bar graph showing the distribution
S of clonogenic cells and LTC-IC within the CD34~ fraction of
circulating CNL cells subdivided according to their high
or low expression of HLA-DR;
Figure 8 is a bar graph showing the distribution
of clonogenic cells and LTC-IC within the CD34+ fraction of
circulating CML cells subdivided according to their uptake
of Rh-123;
Figure 9 is a bar graph showing survival of
circulating CML clonogenic cells and LTC-IC after a brief
exposure to 4-HC;
Figure 10 is a bar graph showing the number of
nonadherent (N~) cells in 5-week old cocultures initiated
with equal numbers of sorted cells seeded onto M2-lOB4
feeders producing human growth factors or human MF is
compared with NA cells in 5-week old cocultures containing
control N2-lOB4;
Figure 11 is a bar graph showing the number of
clonogenic cells in 5-week old cocultures containing M2-
lOB4 cells producing human growth factors or human NF
compared with control M2-lOB4 cells;
Figure 12 is a bar graph showing the number of
LTC-IC as determined by limiting dilution analysis in 5-
week old cocultures initiated with equal numbers of sorted
cells seeded onto human growth factor producing M2-lOB4
cells or human NK compared with the LTC-IC content of 5-
week old cocultures containing control M2-lOB4 cells;
Figure 13 shows the linear relationship between
the number of light density ((1.077g/cm3) T cell-depleted
peripheral blood cells from a representative normal
individual seeded onto pre-established, irradiated normal
marrow feeders and the total number of clonogenic cells
detected when these LTC were harvested and assayed in
methylcellulose 5 weeks later;
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Figure 14 shows limited dilution analysis of
data from a representative experiment in which decreasing
numbers of light density T cell-depleted normal peripheral
blood cells were seeded onto irradiated marrow feeders and
the number of clonogenic cells detectable after 5 weeks
was then determined;
Figure 15 shows bivariate contour histograms of
light density T cell-depleted normal peripheral blood
cells stained with anti-CD34 and anti-HLA-DR;
Figure 16 shows light scatter profiles of T
cell-depleted light density normal blood cells (Panel A)
and the mean + SEM of the percentages of nucleated cells
(open bar), clonogenic cells (stippled bar), and LTC-IC
(solid bar) in each sorted fraction (Panel B);
Figure 17 show a representative histogram of
CD34+, light density T cell-depleted normal blood cells (in
the previously described low SSC window shown in Figure
4A) double-stained with PE conjugated anti-HLR-DR;
Figure 18 show a representative histogram of
CD34~, light density T cell-depleted normal blood cells
double-stained with Rh-123 and sorted into CD34~RH-123
and CD34~RH-123bri9ht fractions (fractions 1 and 2,
respectively; Panel A) and the mean + SEN of the
percentages of nucleated cells (open bar), clonogenic
cells (stippled bar), and LTC-IC (solid bar) in each
sorted fraction are shown in (Panel B) (n=3); and
Figure 19 shows a comparison of the number of
clonogenic cells and LTC-IC surviving a 30 minute exposure
to lOO~g/ml of 4-HC at 37C with 7% erythrocytes present.
DETAILED DESCRIPTION OF THE INVENTION
As hereinbefore mentioned the invention relates to a cell
preparation comprising primitive hematopoietic stem cells
obtained from leukemic patients that are Ph1-positive and
capable of initiating long term culture and producing
clonogenic precursors. The stem cells may be further
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characterized as CD34~ and DR', FSChi~h, SCCIW, and/or Rh~
123~Ve. Preferably the cell preparation has a purity of
circulating primitive leukemic hematopoietic stems cell~
of appro~imately 10~.
The invention further provides a cell preparation
comprising primitive hematopoietic stem cells obtained
from the blood of normal individuals which cells produce
detectable clonogenic progenitors in long term culture.
The primitive hematopoietic stem cells in the preparation
may be further characterized as CD34', and HLA-DR-, FSCLW,
SCC~W, and Rh-123dUll and 4-hydroperoxycyclophosphamide-
resistant with differentiative and proliferative
potentialities similar to LTC-IC in normal marrow.
Preferably the cell preparation has a purity of
circulating primitive hematopoietic stem cells of
approximately 0.5 ~
The primitive hematopoietic stem cells are also referred
to herein as LTC-IC and primitive hematopoietic stem cells
from a patient with leukemia or CML are referred to as
leukemic or CNL LTC-IC. The term LTC-IC used herein refers
to a cell that after a minimum period of 5 weeks in
culture together with certain marrow adherent cells, but
in the absence of exogenous growth factors, produces
detectable clonogenic progenitor cells.
As hereinbefore mentioned the invention also relates to a
method for preparing a cell preparation comprising
primitive hematopoietic stem cells, which are
characterized as Ph1-positive and capable of producing
clonogenic progenitors detectable in long term culture
comprising obtaining a sample from a leukemic patient,
preferably a blood sample from a leukemic patient having
an elevated white blood cell count, most preferably having
a white blood cell count greater than 20 x 109 white blood
cells per litre of blood; and isolating a cell preparation
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from the cell sample which is Philadelphia chromosome
positive and produces detectable clonogenic progenitors in
long term culture. Preferably, the method provides a cell
preparation substantially comprising primitive leukemic
hematopoietic stem cells which are CD34~ HLA-DR+, FSChigh,
SCC~W, and Rh-123~Ve. Preferably, a cell preparation having
a purity of circulating primitive leukemic hematopoietic
stem cells of approximately 10% is obtained.
The cell preparation may be prepared from blood samples of
leukemia patients, preferably from CNL patients having an
elevated white blood cell count, most preferably
containing greater than 20 x 109 white blood cells per
liter. Normal (Ph1-negative) LTC-IC persist in the marrow
of many CNL patients (and to a much lesser extent, normal
clonogenic cells). Blood samples having elevated white
cell counts were found to be a source of cells that are
reproducibly, significantly, and preferentially enriched
for neoplastic progenitors, eliminating the need for
laborious genotyping studies. Thus normal cells, even if
present in such samples at normal levels~ remain well
below the limit of detectability in the methods of the
present invention (as illustrated by the calculations
shown in Table 4). The present inventors have also shown
that more than 97% of LTC-IC from CML patients with high
white blood cell counts and markedly elevated LTC-IC
concentrations exhibit abnormal functional properties
(i.e., self-maintenance) in LTC, consistent with a
leukemic origin.
The present inventors have shown that the primitive Ph1-
positive primitive hematopoietic stem cells differ from
their counterparts in normal individuals with respect to
a number of functionally related properties. The
differences are suggestive of a deregulation in the
control of cell proliferation in CML at the level of the
cells initially responsible for maintenance and expansion
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of the Ph~-positive clone without alteration of their
commitment to, or early differentiation down, each of the
hematopoietic lineages. It is expected that these
findings may provide a useful framework for future
analysis of the mechanism of BCR-ABL-induced multi-lineage
disease.
The majority of both clonogenic cells and LTC-IC in the
circulation of CML patients with high white blood cell
counts are phenotypically similar to one another with
respect to size (FSC), expression of CD34 and HLA-DR,
uptake of Rh-123 and sensiti~ity to 4-HC. In both cases,
the predominant phenotype is that of proliferating or
activated cells (i.e., high FSC, high expression of HLA-
DR, high Rh-123 uptake and relative sensitivity to 4-HC).
However, subtle differences between circulating clonogenic
cells (more activated) and LTC-IC (less activated) in CML
patients are consistently noted. This predominant,
I'abnormal'' phenotype, described herein is essentially the
opposite of that previously shown for the majority of
clonogenic cells and LTC-IC in the circulation of normal
adults, also shared by the majority of LTC-IC in normal
marrow ti.e., low FSC, low expression of HLA-DR, low Rh-
123 uptake and relative insensitivity to 4-HC).
Separation techniques based on one or more of the distinct
phenotypic characteristics of the primitive leukemic
hematopoietic stem cells may be used to isolate the cells
from a sample. A cell preparation comprising primitive
leukemic stem cells may be isolated fro~l a sample based on
the light scattering properties of the primitive leukemic
stem cells. The stem cells may be found in cell fractions
with high FSC and low SSC or fractions with low FSC and
low SSC. The cell preparation may also be isolated based
on other phenotypic characteristics of the stem cells, for
example, their ability to express detectable levels of
HLA-DR or CD34 or their ability to retain Rh-123.
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~enerally, the primitive leukemic hematopoietic stem cells
are CD34+, ~LA-DRhi9h and Rh-123'Ve.
In one embodiment of the invention, a cell preparation
comprisi~g primitive leukemic hematopoietic stem cells may
be isolated by first removing erythrocytes, granulocytes
and platelets from the sample. In particular, a light
density fraction (C1.077 g/cm3) may be isolated by
centrifugation of the blood on Ficoll/Hypaque (FH) to
eliminate the majority of erythrocytes, granulocytes and
platelets. T-cell depletion is generally not required,
since it was found that the number of T cells in initial
CML blood samples was at or below 2~ of the total.
. .
The light density fraction may be fractionated as
generally described below, to obtain a fraction enriched
in CD34'and DR~ cells. In particular, cells, in the light
density fraction may be washed and resuspended in a
physiologic solution, for example Hank's solution with .
fetal calf serum (FCS) containing sodium azide (NaN3)(HFN).
The suspended cells may be stained with an anti-CD34
antibody, for example 8G12, directly conjugated to a
detectable marker, preferably a fluorescent label, most
preferably phycoerythrin (PE) or fluorescein
isothiocyanate (FITC), following the methods described in
Lansdorp et al., J. Exp. Med., Vol. 172, p.363, 1990, then
double-stained with Rhodamine, preferably Rhodamine-123
(RH-123) (Udomsakdi et al., Exp. Hematol, Vol. 19, p. 338,
1991) or antibody specific for HLA-DR conjugated to a
detectable marker, preferably with a fluorescent label,
most preferably anti-HLA-DR-PE may be used (Sutherland et
al., Blood Vol. 74, p. 1563, 1989).
One skilled in the art will appreciate that the detectable
markers conjugated to the HLA-DR and CD34 antibodies
should be selected so as to permit measurement of the
binding of the two antibodies to their respecti~e
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antigens; that is, the detectable markers should be easily
distinguished from each other to facilitate simultaneous
measurement.
The stained, fluorescently labelled cells may be analyzed
and sorted using a fluorescence-activated cell sorter
(FACS) such as the Becton Dickenqon FAC Star Plus.
Fluorescence of Rh-123, FITC and PE-labelled cells may be
measured using 530/30 and 575/26 band pass filters,
respectively, after calibration of the FACS prior to each
sort, preferably with 10 ~m fluorescent beads. Gates may
be set to exclude most of the granulocytes and
erythrocytes using forward light scatter (FSC) and side
scatter (SSC) characteristics as described in Udomsakdi et
al., Exp. Hematol, Vol. 19, p. 338, 1991. Cells appearing
` within this light scatter window (see Figure 5A)
constitute on average 15-20% of the total light density
fraction of CML blood cells. Sorted cells may be
collected in physiological solution, preferably in Hank's
solution with 50% FCS and may be maintained at, for
example 4C until required.
Sorted cells may be used to initiate long term cultures,
by the methods more particularly described below. The
clonogenic progenitor cells resulting from long term
culture may be assayed for clonogenic erythropoietic (BFU-
E), granulopoietic (CFU-GM), and multilineage (CFU-GEMM)
progenitors as described below.
The cell preparation comprising primitive leukemic
hematopoietic stem cells of the invention may also be used
to characterize the differentiative potential of CML LTC-
IC. The relative numbers of different types of clonogenic
progenitors present in 5 week-old LTC ( see discussion
below) provides a consistent average overall measure of
the differentiative behaviour of LTC-IC assayed under
standard LTC conditions. To assess whether this parameter
...- . . ... ...
... , :
20g~25S
- 18 -
is altered in the LTC-IC present in CML blood, the ratio
of BFU-E, CFU-GM and CFIJ-GEMM numbers before and after LTC
of light density CML blood cells may be assessed.
As hereinbefore mentioned the invention further provideR
a method for preparing a cell preparation comprising
primitive hematopoietic stem cells obtained from the blood
of normal individuals which cells produce detectable
clonogenic progenitors in long term culture comprising
obtaining a blood sample from a normal individual, and
isolating a cell preparation from the cell sample which
contains cells which produce detectable clonogenic
progenitors in long term culture. Preferably, the cell
- preparation obtained substantially comprises primitive
hematopoietic stem cells which are CD34' and HLA-DR-,
FSC~W, SCC~W, Rh-123dULL and 4-hydroperoxycyclophosphamide-
resistant with differentiative and proliferative
potentialities similar to LTC-IC in normal marrow.
Preferably, a cell preparation having a purity of
circulating primitive hematopoietic stem cells of
approximately 0.5 to 1% is obtained.
.~ .
Separation techniques based on one or more of the distinct
phenotypic characteristics of the primitive hematopoietic
stem cells may be used to isolate the cells from a blood
sample. A cell preparation comprising primitive stem cells
25 - may be isolated from a sample based on the light
scattering properties of the primitive stem cells. The
stem cells may be found in cell fractions with low FSC and
low SSC. The cell preparation may also be isolated based
on other phenotypic characteristics of the stem cells, for
example, their ability to express detectable levels of
HLA-DR or CD34 or their ability to retain Rh-123.
Generally, the primitive leukemic hematopoietic stem cells
are CD34~, HLA-DR-Ve and Rh-123dU~L. Specific protocols for
isolating LTC-IC from the blood of normal individuals are
set forth in the examples herein.
.;,., ~ .
-: - .
- ~ ~
~ '
208025~
-- 19 --
As hereinbefore mentioned, the invention provides a method
for quantitating primitive leukemic hematopoietic stem
cells in patients with leukemia comprising obtaining a
cell sample which contains primitive lsukemic
hematopoietic stem cells from a leukemic patient;
optionally enriching primitive leukemic hematopoietic stem
cells in the sample; coculturing the sample with a feeder
cell layer for at least five weeks under conditions which
permit the production of clonogenic progenitor cells;
harvesting nonadherent cells and adherent cells;
subjecting the harvested cells to secondary assay culture
under suitable conditions to express clonogenic progenitor
cells; detecting and quantitating the number of clonogenic
progenitor cells; and, quantitating the number of
primitive hematopoietic stem cells in the sample on the
basis of the linear relationship between the number of
clonogenic progenitor cells and the number of primitive
leukemic hematopoietic stem cells initially in the sample.
The above method may be used to ~uantitate CML LTC-IC.
The above method of the invention may be carried out on
heparinized cell samples obtained from the bone marrow or
blood, preferably the peripheral blood, of a mammal,
preferably a human. In a preferred embodiment, the cell
sample may be obtained from a patient with CML, having an
- 25 elevated white blood cell count, preferably of >20 x 104
white blood cells per litre of blood. As discussed above
such samples are substantially devoid of normal LTC-IC
hematopoietic stem cells and are enriched in leukemic LTC-
IC hematopoietic stem cells. Thus it is not necessary to
distinguish normal and leukemic LTC-IC as is required for
bone marrow cell samples, as described below.
The cell sample may be pre-treated prior to coculturing
with the feeder layer to enrich primitive leukemic
hematopoietic stem cells in the sample. Red blood cells
may be removed from the cell sample, for example by a
... .
.': ' . .
.
20802~
- 20 -
brief exposure to ammonium chloride. For blood cell
samples, a preparation of light density cells ~for example
<1.077 gm/cm3) may be isolated by centrifugation on Ficoll-
Mypaque. Preferably, where the initial cell sample is
obtained from the blood of a mammal without an elevated
white blood cell count, the sample may be depleted of T
cells. T cell depletion may be effected, for example by
incubation of the light density cells with 2-amino-
ethylisothiouronium bromide-treated sheep erythrocytes for
30 minutes on ice followed by further centrifugation to
remove the rosetted T cells as described generally by
Marsden et al., J. Immunol. Nethods, Vol. 33, p. 323,
1980. T cell removal prevents the spontaneous activation
and outgrowth in vitro of Epstein-virus transformed B
lymphocytes. Since the T cell content of the high white
blood cell count peripheral blood specimens obtained from
CML patients is already decreased to a few percent,
further removal of T cells from these samples may not be
necessary.
An enriched blood sample which is CD34' and DR~ may also be
used in the method. Such an enriched sample may be
obtained by treating the blood sample with antibodies to
CD34 labeled with a detectable marker and antibodies to
HLA-DR labeled with a detectable marker, wherein the
antibodies to CD34 and antibodies to HLA-DR are labeled
with different detectable markers, and isolating an
enriched sample which is CD34+ and DR~ by means of the
labels. Preferably the detectable marker is a fluorescent
label, most preferably phycoerythrin (PE) or fluorescein
isothiocyanate (FITC) (Lansdorp et al., J. Exp. Med., Vol.
172, p.363, 1990).
The samples or enriched samples are cocultured with a
feeder cell layer for at least five weeks under conditions
which permit the production of clonogenic progenitor
cells. Examples of suitable feeder cells include
.~. ..
- ~
~ ', - ~ ' .
208û2~5
-- 21 --
irradiat~d normal marrow adherent cell layers, subcultured
from primary LTC. Sutherland et al., Blood, Vol. 74, p.
1563, 1989 provide a discussion of suitable feeder layers.
It will be appreciated that other adherent cells, such as
murine fibroblasts may be used as the feeder cell layer.
The culture conditions are as generally described in Eaves
et al., J. Tissue Culture Methods, Vol. 13, p. 55, 1991.
In particular the culture may be maintained at
physiological temperatures, preferably in the range from
30-40C, preferably 32-37C. In a preferred embodiment the
cultures are maintained at 37C for the first 3-4 days and
then subsequently at 30-35C, most preferably 33C. P.fter
the first 7 to 10 days half of the medium and half of the
nonadherent cells may be removed and replaced with new LTC
medium. This feeding procedure may then be repeated
periodically, preferably after 14 days and at weekly
intervals thereafter.
. . .
After at least 5 weeks, nonadherent cells and adherent
cells may be harvested. The adherent cells may be
harvested using known techniques for example using
trypsin.
The harvested cells are subjected to secondary assay
culture under suitable conditions to express clonogenic
progenitor cells and the number of clonogenic progenitor
cells are detected and quantitated. The secondary assay
culture may be a semi-solid assay culture, for example a
methylcellulose culture, and erythropoietic (BFU-E),
granulopoietic (CFU-GN) and multi-lineage (CFU-GENN)
progenitors may be detected and quantitated. Preferably,
the assay is performed in a standard methylcellulose
culture containing human erythropoietin and agar-
stimulated human peripheral leukocyte conditioned medium
(Terry Fox Laboratory Media Preparation Service,
Vancouver, BC~. Harvested cells may be plated at
.
'. " . ;' ' ' ' : ~ , ~
2080255
- 22 -
appropriate concentrations preferably 5 x 105 cells per 1.1
ml assay, in replicate methylcellulose cultures under
standardized conditions optimized for expression of the
colony-forming potential of these cells as assessed after
18-21 days incubation at 37C using previously described
colony scoring criteria (Cashman et al., Blood, Vol. 66,
p. 1002, 1985).
The methodology for colony generation and criteria for
colony recognition are generally as described in Coulombel
et al., Blood, Vol. 62, p. 291, 1983. Total clonogenic
cell numbers refers to the sum of BFU-E, CFU-GM, CFU-GENM
detected in direct assays using these procedures.
Where the original cell sample may be contaminated with
normal primitive hematopoietic stem cells, primitive
leukemic hematopoietic stem cells may be identified by
genotyping the colonies produced in the secondary culture.
Genotyping may be carried out by cytogenetic analysis of
individually removed single or small pools of colonies as
generally described in Fraser et al., Cancer Genet.
Cytogenet., Vol. 24, p.1, 1987, to allow the proportion of
normal and leukemic clonogenic cells to be determined.
Where CML bone marrow cell samples are used in the method
of the invention, it is necessary to perform cytogenic
analysis on the colonies produced from the clonogenic
progenitors to distinguish Phl-positive and Ph1-negative
LTC-IC. In contrast to CML blood, Ph1-positive primitive
leukemic hematopoietic stem cells would be expected to
represent a minority population relative to normal LTC-IC
in CML marrow (Oster et al., Blood (abstr) Vol. 70, p.
266a 1987 - Barnett et al., Bone Narrow Transplant, Vol.
4, p. 345, 1989). The concentration of Ph1-positive LTC-IC
... . . .
.
.- ~
-- :
- 23 - 2080255
(relative to other nucleated cells) in CML marrows has
been found to be quite variable and, in general, is
markedly reduced, both by comparison to LTC-IC values in
control marrows and by comparison to normal (Ph1-negative)
LTC-IC co-existing in the same CML marrow.
The relative number of primitive hematopoietic stem cells
in the sample is quantitated on the basis of the linear
relationship between the number of clonogenic progenitor
cells and the number of primitive leukemic hematopoietic
cells cocultured with the feeder cell layer.
It is possible to quantitate absolute primitive leukemic
hematopoietic stem cells in the sample by employing
limiting dilution analysis in the method of the invention.
For such methods, samples containing varying
concentrations of primitive leukemic hematopoietic stem
cells, for example, 100 ~1 aliquot samples containing from
50 to 2 x 105 light density cells may be cocultured with a
feeder cell layer and the harvested cells may be plated in
methylcellulose assay cultures to enable detection of one
or more clonogenic cells in each sample. From the
proportion of positive and negative primitive leukemic
hematopoietic stem cells defined in this way, the
frequency of primitive leukemic hematopoietic stem cells
in different input samples may be calculated preferably
using Poisson statistics (Taswell, C., J. Immunol. Vol.
126, p. 1614, 1981 - Coller et al., Methods in Enzymology,
Vol. 121, p. 412, 1986).
The proliferative potential of the primitive hematopoietic
stem cells as indicated by the average 5 week output of
clonogenic cells per primitive leukemic hematopoietic stem
cells may then be derived in each case. Knowledge of the
5 week clonogenic cell output per primitive leukemic
hematopoietic stem cells allows absolute values to be
derived from total 5 week clonogenic cell yields measured
. .
. , - . .
: , - :' .' .:
208~25~
- 24 -
in cul~ures initiated with non-limiting inocula.
Primitive leukemic hematopoietic stem cells values may
thus be obtained for samples, and the concentration of
primitive leukemic hematopoietic stem cells per ml of
blood then calculated assuming 100~ LTC-IC recovery in the
light density fraction assayed (Sutherland et al., Blood,
Vol. 74, p. 1563, 1989).
.
The present inventors have demonstrated that, on average,
primitive leukemic hematopoietic stem cells circulate at
a 10-fold lower frequency than clonogenic cells although
these two parameters showed a highly significant
association. By comparison the ratio of circulating
primitive leukemic hematopoietic stem cells to clonogenic
cells in normal blood appears to be much lower.
.
Primitive CML hematopoietic stem cells were found to
produce on average, a similar number of clonogenic cell
progeny after 5 weeks cocultured with a feeder cell layer
as do their normal counterparts in the blood or marrow of
normal individuals. However, a number of abnormalities in
the primitive CML hematopoietic stem cell population were
also revealed. First, their distribution between marrow
and blood was shown to be grossly altered, even more
dramatically than is the case for Ph1-positive clonogenic
cells. Both populations increase exponentially in the
blood with linear increases in the white blood cell count,
but Ph1-positive primitive hematopoietic stem cells appear
to be present at relatively reduced frequencies in CNL
marrow whereas Ph1-positive clonogenic cell frequencies in
CNL marrow are relatively normal (Eaves et al., Exp.
Hematol., Vol. 8, p. 235, 1980).
In human long term cultures, primitive normal clonogenic
cells in the adherent layer alternate weekly between a
quiescent and a dividing state (Cashman et al., Blood,
Vol. 66, p. 1002, 1985) and in murine long term cultures,
~.
20~02~
- 25 -
it has been possible to demonstrate that extensive
proliferation of some long-term totipotent reconstituting
cells does occur. In long term cultures initiated with
primitive leukemic hematopoietic stem cells, their
derivative primitive clonogenic progeny divide
continuously suggesting a defective but unregulated
mechanism for inevitable expansion of the Ph~-positive
clone. Thus the method of the invention may serve as an
important model for further dissection of the mechanisms
that regulate normal versus CNL recovery patterns in vivo.
As hereinbefore mentioned, the invention also relates to
the use of the above described method of the invention in
the diagnosis of CML and in characterizing the disease
state of a patient with leukemia by quantitating primitive
leukemic hematopoietic stem cells in the blood and bone
marrow of a patient. The efficacy of various treatments
for leukemia, including chemotherapy, radiation therapy
and bone marrow transplants may be assessed using the
method of the invention by determining the number and
characteristics of primitive hematopoietic leukemic stem
cell~ in the blood and bone marrow of the patient at time
periods before and after treatment.
Thus, the quantitative assay of the invention for a
primitive leukemic hematopoietic stem cell population
facilitates a variety of studies to further characterize
these cells, obtain a better estimate of the number of
leukemic stem cells in individual leukemia patients, and
to devise more effective treatment strategies both in and
ex vivo.
As hereinbefore mentioned the invention further relates to
a method for quantitating primitive hematopoietic stem
cells in blood samples of normal individuals comprising
obtaining a blood sample which contains primitive
hematopoietic stem cells from a normal individual,
:: ~
-
: ., .
.
: . , :.. : : - :
208025~
- 26 -
optionally enriching primitive hematopoietic stem cells in
the blood sample; coculturing the sample with a feeder
cell layer for at least five weeks under conditions which
permit the production of clonogenic progenitor cells;
harvesting ncnadherent cells and adheren$ cells;
subjecting the harvested cells to secondary assay culture
under suitable conditions to express clonogenic proqenitor
cells; detecting and quantitating the number of clonogenic
progenitor cells; and, quantitating the number of
primitive hematopoietic stem cells in the blood sample on
the basis of the linear relationship between the number of
clonogenic progenitor cells and the number of primitive
hematopoietic stem cells initially in the sample.
The blood samples from normal individuals may be blood
samples containing peripheral blood mononuclear cells
obtained from normal individuals as a byproduct of
plateletphereses. Samples may be depleted of T cells by
incubation with 2-aminoethylbromide isothiournium-treated
sheep red blood cells and subsequent isolation of a light
density fraction after centrifugation on Ficoll-Hypaque.
An enriched blood sample from a normal individual which is
CD34' may also be used in the method. Such an enriched
sample may be obtained by treating the blood sample with
antibodies to CD34 labeled with a detectable marker and
isolating an enriched sample which is CD34~by means of the
labels. Preferably the detectable marker is a fluorescent
label, most preferably phycoerythrin (PE) or fluorescein
isothiocyanate (FITC) (Lansdorp et al., J. Exp. Med., Vol.
172, p.363, 1990).
The blood sample or enriched blood sample may be
cocultured with a feeder cell layer to produce clonogenic
progenitor cells and the clonogenic progenitor cells may
be detected and quantitated using the procedures outlined
above. The number of primitive hematopoietic stem cells in
the blood sample is determined on the basis of the linear
'
208025~
- 27 -
relationship between the number of clonogenLc progenitor
cells and the number of primitive hematopoietic stem cells
initially in the sample as discus-~ed previously.
The invention further relates to a system for testing for
a substance that affects hematopoiesis of primitive
hematopoietic stem cells comprising: preparing a cell
suspension that is enriched in primitive hematopoietic
stem cells; coculturing the cell suspension with a feeder
cell layer for at least 5 weeks in the presence of a
substance which is suspected of affecting the
hematopoiesis of primitive hematopoietic stem cells and
assessing LTC-IC maintenance, clonogenic cell production,
and/or production of nonadherent cells.
The cell suspension may comprise primitive hematopoietic
stem cells obtained from the blood of normal individuals.
The cell suspension may also be obtained from blood or
bone marrow of leukemic patients, preferably from the
blood of a leukemic patient having an elevated white blood
cell count and the primitive hematopoietic stem cells may
be Philadelphia chromosome positive and produce detectable
clonogenic progenitors in long term culture. Preferably
the cell suspension is highly enriched in primitive
hematopoietic stem cells. Methods for pre-treating samples
to obtain enriched preparations of primitive hematopoietic
stem cells have been described above.
The substance may be added to the culture of the cell
suspension and feeder cell layer, or the feeder cell layer
may be genetically engineered to express the substance
i.e. the feeder cell layer may serve as an endogenous
source of the substance. Nurine marrow-derived stromal
cell lines such as N2-lOB4, may be engineered by
retroviral-mediated gene transfer to produce specific
substances such as specific human factors. Examples of
specific human growth factors which can be produced using
. .
; ~' , " :
- ~ ' :, ` ~ ::
.: . .
' -
~ .
208025~
- 28 -
the engineered feeder cell layer are G-CSF, GM-CSF, and
interleukins such as IL-3, IL-4 and IL-6. The engineered
cell feeder layer is constructed and maintained such that
it releases the substance into the medium for the desired
period of time for the long term culture. Through the use
of genetically engineered growth factor-producing feeders
it may be possible to reproduce the way in which regulator
substances may be localized in the adherent layer and/or
presented to adjacent hematopoietic cells on the
assumption that this might influence the nature and
magnitude of their effects. The feeder cell layer may be
human marrow stromal cells which may be induced to produce
human factors such as G-CSF and GN-CSF.
The system may be used to analyze the affects of
substance(s) on different stages of hematopoiesis.
Effects on cells at very early, intermediate, and late
stages of hematopoiesis may be evaluated by assessing the
number of clonogenic cell precursors, clonogenic cells,
and mature granulocyte and macrophage progeny present in
the cultures after 5 weeks.
LTC-IC maintenance, clonogenic cell production and
production of nonadherent cells may be assessed using the
methods described herein.
The present inventors have surprisingly found that CML
LTC-IC are selectively disadvantaged in long term culture
compared to normal LTC-IC. In particular, the present
inventors have shown that, in spite of a normal output of
clonogenic cell progeny by Ph1-positive LTC-IC and the
provision of a pre-established feeder layer derived from
a normal marrow donor, their initial maintenance in the
- ' '
20~025~
- 29 -
LTC system was highly compromised relative to normal LTC-
IC. The beh~viour of normal and leukemic LTC-IC in the
LTC may indicate how these cells behave in vivo under
analogous conditions of stimulation.
Accordingly, the invention further relates to a method of
purging a mammalian bone marrow sample containing
primitive leukemic hematopoietic stem cells to prepare a
bone marrow cell suspension substantially free of
- primitive leukemic hematopoietic stem cells comprising
culturing the bone marrow sample in long term culture in
vitro. It will be appreciated that such a purging method
will be useful for removing leukemic precursor cells from
a bone marrow sample to be introduced into a human
recipient, for example in the case of autologous bone
marrow transplan~ for a leukemia patient. The preparation
of samples and the long term culture procedure have been
previously described herein.
The following non-limiting examples are illustrative of
the present invention.
EXA~PLES
Example 1
Preparation of Cell Samples
Heparinized bone marrow (BN) aspirate cells were obtained
with informed consent from normal individuals and Ph1-
positive CML patients undergoing marrow harvests for
transplantation. Heparinized normal blood from additional
normal individuals and CNL blood from CML patients
undergoing routine hematologic assessment was similarly
obtained. All CML patients were Ph1-positive and in stable
chronic phase. For the initial experiments with CNL
blood, only samples containing >20 x 109 white blood cells
(WBC) per L were used, as this allowed selection of
patients whose circulating Ph1-positive progenitors were
sufficiently elevated that even after maintenance in LTC
only Ph1-positive cells were detected (see Figure 3A) thus
t , ~
- .,
' ~
20~025~
- 30 -
avoiding the necessity for confirmatory progenitor
genotyping as is required for similar experiments with CML
BM .
BM cells for initiation of LTC were either used without
further processing or after removal of contaminating red
blood cells when the nucleated cell count in the original
specimen was less than 2 x 107 cells/ml. For clonogenic
cell assays, red cells were first lyzed by brief exposure
to ammonium chloride generally as described in Turhan et
al., N. Engl. J. Med., Vol. 320, p. 1655, 1989, and the
cells then washed twice in Iscove's medium plu6 2~ fetal
calf serum. For blood cell samples, light density (<1.077
gm/cm3) cells were isolated by centrifugation on Ficoll-
Hypaque either with (normal blood) or without ( CML blood)
T cell-depletion. This involved incubation of the light
density cells with 2-aminoethylisothiouronium bromide-
treated sheep erythrocytes for 30 minutes on ice followed
by further centrifugation at 4C on Ficoll-Hypaque to
remove the rosetted T cells, generally as described in
Marsden et al., J. Immunol. Methods, Vol. 33, p. 323,
1980. These were reduced, on average, by this procedure
by more than 98~ according to FACScan analysis of CD2-
positive cells in a lymphocyte gated population. The
primary purpose of T cell removal was to prevent the
spontaneous activation and outgrowth in vitro of Epstein-
virus transformed B lymphocytes which preliminary
experiments showed occurs regularly within 5 weeks when
inadequately T cell--depleted normal peripheral blood
samples were co-cultured with irradiated marrow adherent
layers. Since the T cell content of the high WBC count
peripheral blood specimens obtained from CML patients was
already decreased to a few percent, further removal of T
cells from these samples was not undertaken and
development of spontaneous transformants was not
encountered.
:~
2~80255
- 31 -
Example 2
Long-Term Cultures (LTC)
Test cells, prepared as described in Example 1, were
cultured in LTC medium generally as described in Eaves et
al., J. Tissue Culture Methods, Vol. 13, p. 55, 1991, at
varying initial cell concentrations in dishes or wells
containing (or not containing) a standardized number of
irradiated (15 gy) normal marrow adherent layer cells
subcultured from primary LTC (Sutherland et al., Blood,
Vol. 74, p. 1563, 1989) according to the particular
experimental design. All LTC were maintained at 37C for
the first 3-4 days and then subsequently at 33C. After
the first 7 to 10 days half of the medium and half of the
nonadherent cells were removed and replaced with new LTC
medium. This feeding procedure was then repeated after
14 days and at weekly intervals thereafter. For all LTC-
IC assays, cultures were initiated on normal marrow feeder
layers, as described above, and then maintained for 5
weeks. At this time all of the nonadherent cells and the
trypsinized adherent cells were harvested, washed and
plated in methylcellulose cultures for quantitation of
clonogenic cells as described below.
For LTC-IC maintenance studies, the entire contents of
primary LTC were harvested at the times indicated,
aliquots used to initiate secondary LTC on new irradiated
feeders and these secondary LTC were then maintained a
further 5 weeks prior to harvesting and plating of the
cells in methylcellulose assays. The number of clonogenic
cells detected at this time provided a relative measure of
the LTC-IC in the primary LTC at the time they were
harvested. This value was then normalized by the number
of LTC-IC detected in primary LTC-IC assays of the
original cell suspension, to yield a percent input value.
Example 3
Colony Assays
, . .
.-, ~
..
: ; -~ ~ - . .:
~ - . ' - - ~ .
2080255
- 32 -
Erythropoietic (BFU-E), granulopoietic (CFU-GN and multi-
lineage (CFU-GEMM) progeni~ors were assayed by plating
test cell suspensions at appropriate concentrations in
replicate methylcellulose cultures under standardized
conditions optimized for expression of the colony-forming
potential of these cells as assessed after 18-21 days
incubation at 37C using previously described colony
scoring criteria (Cashman et al., Blood, Vol. 66, p. 1002,
1985). In all assays of fresh or cultured CML marrow,
colonies produced in methylcellulose were genotyped by
cytogenetic analysis of individually removed single or
small pools of colonies (Fraser et al., Cancer Genet.
Cytogenet., Vol. 24, p.1, 1987) to allow the proportion of
normal and leukemic clonogenic cells, and hence LTC-IC, to
be determined.
E~ample 4
CNL LTC-IC ~SSAYS
The validity of using the clonogenic cell producing
property of LTC-IC as an endpoint for their quantitation
depends on the existence of a linear relationship between
the number of LTC-IC seeded into the cultures and the
number of clonogenic cells present 5 weeks later
regardless of the input LTC-IC concentration. This was
previously demonstrated for assays of LTC-IC in normal
marrow cell suspensions (Sutherland et al., Proc. Natl.
Acad. Sci., Vol. 87, p. 3584, 1990).
Previous studies had shown that LTC initiated with
peripheral blood cells from CML patients with high WBC
counts (and a marked elevation in circulating Ph1-positive
progenitors, (Eaves et al., Bailliere's Clinical
Haematology Vol. 4, pg. 931, 1990)) when analyzed 4-8
weeks later contained high numbers of exclusively Ph1-
positive clonogenic progenitors (Eaves et al., Proc. Natl.
Acad. Sci., Vol. 83, p. 5306, 1986) in contrast to LTC
initiated with CML BM (Coulombel et al., N. Engl. J. Med.,
.
,
. - . .
. ". ~.
-`, .
.~ .
.
20802~
- 33 -
Vol. 308, p. 1493, 1983). This suggested that it might be
possible to use CML peripheral blood from such patients as
an enriched source of primitive leukemic cells to
investigate the relationship between cell input and
S leukemic clonogenic cell output 5 weeks later.
In a series of experiments using cell samples, prepared
from the peripheral blood from 8 different CML patients
with high WBC counts, in which the number of light density
peripheral blood input cells was varied from 5 x 103 to a
maximum of 107 cells per 2.5 ml LTC (in 35 mm tissue
culture dishes), the slope of the line relating the
logarithm of the inoculum size (total nucleated cells) to
the logarithm of the number of clonogenic cells detected
: after these LTC had been maintained for 5 weeks was l.05
lS + 0.21 (which is not significantly different from l.0,
p~0.5).
The results for a representative patient are illustrated
in Figure l, and show that the conditions described for
the LTC-IC assay in Example 2 are suitable for the
detection and quantitation of Ph1-positive LTC-IC. Figure
l demonstrates the linear relationship between the number
of light density CML peripheral blood cells seeded into
individual LTC (containing irradiated pre-established
normal marrow feeders) and the number of clonogenic cells
detected in secondary methylcellulose assays of cells
harvested from these primary LTC 5 weeks after their
initiation. Each point represents a single LTC. All
points are derived from a single representative experiment
using cells from a CML patient with a WBC count of l90 x
: 30 109/L. The slope of the regression line fitted to this
data set is 0.81+0.13.
.,
Example S
: Measurements of leukemic LTC-IC in CML blood and marro~.
Because the relative output of clonogenic cells from
:: ~ . . : : . . . :
. . . ~ : . .
. . . :
- . - : -
- . ,:
20802~
- 34 -
circulatinq leukemic LTC-IC was found to be constant under
the assay conditions used even down to limiting numbers of
input cells, as shown, for example, in Figure 1,
quantitation of absolute leukemic LTC-IC numbers by
limiting dilution analysis was possible. For such
experiments, blood from CNL patients with elevated WBC
counts was again used as a highly and selectively enriched
source of leukemic LTC-IC. Irradiated normal marrow
derived feeders were subcultured into 96 well flat bottom
Nunclon plates (Sutherland et al., Proc. Natl. Acad. Sci.,
Vol. 87, p. 3584, 1990) and then from 50 to 2 x 105 light
density cells added per well in volumes of 1~0 ~1 with 23
+ 1 wells per group. Five weeks later, all of the cells in
each well were suspended and plated in methylcellulose
assay cultures to enable detection of one or more
clonogenic cells per well. From the proportion of
positive and negative LTC defined in this way, the
frequency of LTC-IC in 6 different input samples was
calculated using Poisson statistics (Taswell, C., J.
Immunol. Vol. 126, p. 1614, 1981 - Coller et al., Nethods
in Enzymology, Vol. 121, p. 412, 1986).
Results for a representative experiment are shown in
Figure 2. Figure 2 shows limiting dilution analysis of
data from a representative experiment in which decreasing
numbers of light densi~y CML peripheral blood cells (from
a patient with a WBC count of 21 x 109/L) were seeded onto
irradiated marrow feeders and the cultures then assayed 5
weeks later for the presence (positive cultures) or
absence (negative cultures) of 21 clonogenic cell. In
this experiment, the frequency of LTC-IC in the suspension
assayed (i.e., the reciprocal of the concentration of
cells that gave 37~ negative cultures) was 1 per 7.6 x 104
cells (95% confidence limits = 1 per 5.3 x 104 - 1 per 11.0
X 104).
The average 5 week output of clonogenic cells per Ph1-
. . ; -
.
.
-
208025~
- 35
positive LTC-IC was then derived in each case. The
results of this latter calculation are shown in Table 1
together with results obtained when the same procedure and
assay conditions were used to analyze LTC-IC in normal
marrow or blood. The proliferative potential o~ all thase
types of LTC-IC as assessed by this 5 week clonogenic cell
output endpoint can be seen to be both similar and
relatively constant, providing further support for the use
of the LTC-IC assay to quantitate and charac~erize a very
primitive Ph1-positive cell type.
Knowledge of the 5 week clonogenic cell output per
leukemic CML allows absolute values to be derived from
total 5 week clonogenic cell yields measured in cultures
initiated with non-limiting inocula, which are
experimentally easier to perform than limiting dilution
analyses. LTC-IC values were thus obtained for peripheral
blood samples from an additional 20 CML patients, and the
concentration of LTC-IC per ml of blood then calculated
assuming 100% LTC-IC recovery in the light density
fraction assayed (Sutherland et al., Blood, Yol. 74, p.
1563, 1989). The results are shown in Figure 3 together
with circulating LTC-IC values obtained from similar
measurements of T-depleted, light density peripheral blood
samples from a large series of normal individuals.
Figure 3 shows the LTC-IC concentration (per ml) in the
peripheral blood of different CML patients (solid circles)
as compared to 23 normal individuals (the open circle in
each panel shows the mean + SEM of 2.9 + 0.5 LTC-IC per ml
measured in these individuals) as a function of the WBC
count (per ml) (Panel A), or the peripheral blood
clonogenic progenitor tBFU-E + CFU-GM + CFU-GENM) content
per ml (Panel B). Absolute LTC-IC values were obtained
either directly by limiting dilution analysis, or
indirectly from the total clonogenic cell output measured
at week 5 divided by the average number of clonogenic
~,: . . - ~ : -
: . .~ : . . -
2~802~5
cells produced per LTC-IC; i.e., 3 and 4 for CML and
normal LTC-IC, respectively, as described above. A
significant association ~etween the two parameters
measured in Panel B is indicated by a Spearmann's rank
correlation coefficient rS=0.77 (p~0.000l).
In Figure 3A, LTC-IC concentrations in CML blood are
plotted as a function of the W~C count. It can be seen
that LTC-IC numbers increase exponentially such that
values >105-fold higher than normal circulating LTC~IC
levels are seen in patients with the largest tumor
burdens. In Figure 3B, the number of circulating LTC-IC
in individual CML patients is plotted as a function of the
number of circulating clonogenic cells ( BFU-E plus CFU-GM
plus CFU-GEMM per ml) in the same patient. On average,
leukemic LTC-IC were found to circulate at a l0-fold lower
frequency than clonogenic cells although these two
parameters showed a highly significant association
(Spearman's rank correlation coefficient, rS=0.77,
p<0.000l, n=26). By comparison the ratio of circulating
LTC-IC to clonogenic cells in normal blood appeared much
lower (~1:80).
LTC-IC assays were also performed using CML marrow
samples. However, in each of these experiments,
cytogenetic analyses were performed on the colonies
produced from the clonogenic progenitors present after 5
weeks in LTC to distinguish Ph1-positive and Ph1-negative
LTC-IC as, in contrast to CNL blood, Ph1-positive LTC-IC
would be anticipated to frequently represent a minority
population relative to normal LTC-IC in CML marrow. The
concentration of Ph1-positive LTC-IC (relative to other
nucleated cells) in the 12 CML marrows analyzed was quite
variable and in general markedly reduced, both by
comparison to LTC-IC values in control marrows (i.e. '2.8
+ l.4 Ph1-positive LTC-IC per 106 CML marrow cells as
compared to 5S + 12 LTC-IC per 106 marrow cells ~rom normal
'
20~0255
individuals, n=13), and by comparison to normal (Ph1-
negative) LTC-IC co-existin~ in the same CML marrows
tested (for which a Yalue of 5.4 + 1.2 per I06 cells was
obtained).
In summary, Ph1-positive LTC-IC were found to produce on
average, a similar number of clonogenic cell progeny after
5 weeks in LTC as do their normal counterparts in the
blood or marrow of normal individuals. However, a number
of abnormalities in the CML LTC-IC population were also
revealed. First, their distribution between marrow and
blood was shown to be grossly altered, even more
dramatically than is the case for Ph~-positive clonogentic
cells. Both populations increase exponentially in the
blood with linear increases in the WBC count, but Ph1-
positive LTC-IC appear to be present at relatively reduced
frequencies in CNL marrow whereas Ph1-positive clonogenic
cell frequencies in CML marrow are relatively normal
(Eaves et al., Exp. Hematol., Vol. 8, p. 235, 1980).
Second, in spite of a normal output of clonogentic cell
progeny by Ph1-positive LTC-IC and the provision of a pre-
established feeder derived from a normal marrow donor,
their initial maintenance in the LTC system was highly
comprised relative to normal LTC-IC. Whether this is due
to an intrinsic defect in the Ph1-positive LTC-IC that is
not subject to extrinsic modulation and/or whether such
differences may also prevail in vivo have yet to be
determined. ~owever, it is interesting to speculate that
the behaviour of normal and leukemic LTC-IC in the LTC may
indicate how these cells behave in vivo under analogous
conditions of stimulation. One might then expect to see
evidence of a growth advantage of the stem cells in the
Ph1-positive clone in vivo only when most co-existing
normal stem cells were in a quiescent state. The latter
might be anticipated to occur in chronic phase CML
patients managed with conventional therapy, but a
situation more closely resembling that obtained in LTC
.
,, ~ . .
- : ,, ~ :
2080255
- 38 -
might occur in vivo, albeit transiently/ following more
intensive treatment. It is interesting to note that
clinical experience fits well with these predictions (Goto
et al., Blood, Vol. 59, p. 793, 1982; Kantar~ian et al.,
J. Clin. Oncol., Vol. 3, p. 192, 1~85).
Example 6
Diferential maintenance of normal and leu~emic LTC-IC in
culture
Previous studies have found that normal marrow LTC-IC are
well maintained in LTC established from a single input
innoculum (Eaves et al., Effects of Therapy on ~iology and
Kinetics of Residual Tumor, Part A: Pre-Clinical Aspects,
p. 223, 1990; Eaves et al., Ann. N.Y. Acad. Sci. Vol. 628,
p. 298, 1991) and similar kinetics are seen when highly
purified LTC-IC from normal marrow are seeded onto
preestablished feeders (Sutherland et al., Proc. Natl.
Acad. Sci., Vol. 87, p. 3584, 1990). Figure 4 shows the
corresponding results obtained when light density
peripheral blood cells from CML patients with high WBC
counts were seeded onto irradiated human marrow feeders
and the number of LTC-IC were determined by harvesting
these primary LTC and performing secondary LTC IC assays
as described above.
In particular, Figure 4 shows the differential kinetics of
CML (solid symbols) versus normal (open symbols) LTC-IC in
LTC initiated from cells seeded onto irradiated normal
marrow feeders. Values shown are means -~ SEM after
normalization of data in individual experiments by setting
LTC-IC values in the primary inoculum in each experiment
to 100%; n=6 for CML (peripheral blood LTC-IC), n=5 for
LTC-IC in normal blood (open circles) and n=2 for LTC-IC
in normal marrow topen triangles). Open squares show
previously published data for LTC-IC in normal unseparated
marrow cultured in the absence of pre-established feeders
-(Eaves et al., Ann N.Y. Acad. Sci., Vol. 628, p. 298,
: :
.
.,
208025~
- 39 -
1991) .
For comparison, analogous experiments were performed for
primary LTC established by seeding light density, T-
depleted normal peripheral blood or normal marrow buffy
coat cells onto pre-established marrow feeders. Normal
LTC-IC maintenance in such cultures was the ~ame
regardless of the source of LTC-IC with no decrease in
overall population size during the first 10 days. In
contrast, the leukemic LTC-IC population showed an
immediate and rapid rate of decline down to ~3~ of input
values within the same initial period during which time
the cultures had not been manipulated in any way except to
reduce the temperature from 37C to 33C.
The following materials and methods were used in the
studies outlined in Examples 7 to 9:
Cells
Heparinized blood samples were obtained with informed
consent from CNL patients undergoing routine hematologic
assessment. All patients were Ph1-positive and in chronic
phase. As shown in Table 2, the number of circulating
LTC-IC in all patients studied was abnormally elevated (by
a factor of from >400-fold to ~ 105-fold above the average
normal value of ~ 2.9 + 0.5 per mL. The light density
fraction (<1.077 g/cm3) was isolated by centrifugation of
the blood on Ficoll/Hypa~ue (FH) to eliminate the ma~ority
of erythrocytes, granulocytes and platelets and to obtain
a preliminary enrichment of progenitor cells.
Normal blood samples were obtained with informed consent
from normal individuals undergoing platelet/leukapheresis
and from these a light density T cell-depleted fraction
was then isolated by rosetting with sheep erythrocytes and
centrifugation on FH as generally described in Marsden et
al., J. Immunol. Methods., Vol. 33, p. 323, 1980). The
~ - . ; ~ . . ,
- . . - : .
~ , , ' ~ ': , '
: ~: ~ :, . ,:. ,:
208~25~
- 40 -
number of remaining CD2-t (T cells) detected by FACScan
analysis of this T cell-depleted, light density fraction
of normal blood cells represented, on average, <2~ of the
total. Since the number of T cells in initial CML blood
samples was already at or a below this level, the T-cell
depletion step was not performed on CML blood samples.
Heparinized normal marrow aspirate cells were leftovers
obtained with informed consent from allogenic donors
providing marrow for transplantation.
: 10 Staining and Flow Cytometry
Cells were washed twice and resuspended in Hank's solution
with 2% fetal calf serum (FCS) and 0.1% sodium azide
(NaN3)(HFN). Cells were first stained with an anti-CD34
antibody (8G12) directly conjugated to phycoerythrin (PE)
3Or fluorescein isothiocyanate (FITC), following the
methods described in Lansdorp et al., J. Exp. Ned., Vol.
172, p.363, 1990, then double-stained with 0.1 ~g/ml
Rhodamine-123 (RH-123) (Udomsakdi et al., Exp. Hematol,
Vol. 19, p. 338, 1991) or 1-2 ~g of anti-HLA-DR-PE (107
cells/ml) (Sutherland et al., Vol. 74, p. 1563, 1989).
Stained cells were analyzed and sorted using a Becton
Dickinson FACStarPlUs fluorescence-activated cell sorter
(FACS) equipped with an argon laser emitting at 488 nm.
Fluorescence of Rh-123, FITC and PE-labelled cells was
measured using 530/30 and 575/26 band pass filters,
respectively, after calibration of the FACS prior to each
sort with 10 ~m fluorescent beads. In some experiments,
gates were set to exclude most of the granulocytes and
erythrocytes using previously described forward light
scatter (FSC) and side scatter (SSC) characteristics.
(Udomsakdi et al., Exp. Hematol, Vol. 19, p. 338, 1991)
Cells appearing within this light scatter window
constituted on average 15-20% of the total light density
fraction of CML blood cells, as shown in Figure 5A.
Figure 5 shows distribution according to light scatter
characteristics of total cells (representative sample -
.:
. ~ . . .
, , . - . , .
:' , . , '
.
2080255
- 41 -
Panel A), and clonogenic cells (open bar~) and LTC-IC
(solid bars) (combined results for patients - Panel B) in
the light density fraction of CML blood. Results for
fractions I, II and III shown in Panel B are as defined in
Panel A. Error bars in Panel B indicate the mean + lSEM
of values obtained on each of 5 patients studied
individually. Sorted cells were collected in Hank~s
~olution with 50% FCS and were maintained at 4C until
plated.
Functional ~ssay~
Cells from primary blood samples or from LTC harvests were
assayed for clonogenic erythropoietic (BFU-E),
granulopoietic (CFU-GM), and multilineage (CFU-GEMM)
progenitors in standard methylcellulose cultures
containing 3 units per ml of human erythropoietin and 10%
agar-stimulated human peripheral leukocyte conditioned
medium (Terry Fox Laboratory Media Preparation Service,
Vancouver, BC). The methodology for colony generation and
criteria for colony recognition were generally as
descried in Coulombel et al., Blood, Vol. 62, p. 291,
1983. Total clonogenic cell numbers refers to the sum of
BFU-E, CFU-GM, CFU-GEMM detected in direct assays using
these procedures. LTC-IC assays were performed by seeding
an aliquot of the test cell suspension into cultures
containing a feeder layer of irradiated (1500 cGy) normal
(allogenic) marrow cells (3 X 104 cells per cm3). These
were subcultured from the adherent layer of previously
established 2-4 week old LTC. (Eaves et al., Proc. Natl.
Acad. Sci. USA, Vol. 83, p. 5306. 1986, Sutherland et al.,
Proc. Natl. Acad. Sci. USA, Vol. 87, p. 3584, 1990). LTC
were initially maintained for 3-5 days at 37C, then
; switched to 33C thereafter. They were then fed weekly by
replacement of half of the growth medium (an enriched ~-
medium containing 12.5% horse serum, 12.5% fetal calf
serum, 10-4 M 2-mercaptoethanol and 10-6 M hydrocortisone)
containing half of the nonadherent cells, with fresh
.
.
. .. . .
.
2~802~5
- 42 -
growth medium.
After a total of 5 weeks, or as specified, the nonadherent
cells were removed, washed, and combined with cells from
those harvested from the trypsinized adherent layer.
These harvested LTC cells were then assayed for clonogenic
cells in standard methylcellulose cultures at an
appropriate concentration (usually 5 x 104 or 105 cells per
l.1 ml assay). The total number of clonogenic cells (i.e.
BFU-E plus CFU-GN plus CFU-&EMM) present in 5 week-old LTC
provides a relative measure of the number of LTC-IC
originally present in the test suspension, as described
above. In some cases absolute LTC-IC values were
calculated by dividing this number by 3, which is the
average output of clonogenic cells per leukemic LTC-IC as
shown above by limiting dilution analysis.
~xample 7
Phenotype Analysis of CML LTC-IC
To determine the light scattering properties of
circulating clonogenic cells and LTC-IC in patients with
CML, light density blood cells were sorted into three
fractions (as illustrated in Figure 5A) and the results
compared with data for normal BN (Sutherland et al., Vol.
74, p. 1563, 1989) and blood progenitors (Figure 5B).
Most of the nucleated cells (~ 85% in the light density
fraction of CML blood had a high SSC (Fraction III) in
contrast to the light density cells in normal blood where
the proportion of such cells is much lower (<40~, data not
shown). The mean number of clonogenic cells and LTC-IC
recovered in each sorted fraction was determined and
expressed as a percentage of the total number of
progenitors present in the starting (light density) cell
suspension of each sample studied. As shown in Figure 5B,
it can be seen that the majority of both the clonogenic
cells and LTC-IC in CML blood were consistently found in
fraction II (i.e. cells with high FSC but low SSC). Cells
:
.:
,'
: . - :::: .. . .
:: -
20~2~
- 43 -
from this fraction also generally produced more nucleated
cells (as well as clonogenic cells) after 5 weeks in LTC
(both in the adherent and nonadherent layer) than other
fractions on a per cell basis. However, a significant
proportion of the circulating CML clonogenic cells (~ 15%)
and LTC-IC (~ 30~) were detected in a population
characterized by low FSC and low SSC (fraction I). Some
circulating C~L clonogenic cells (~ 5%) were found amongst
the cells with a high SSC (fraction III). These findings
suggest subtle differences between circulating C~L
clonogenic cells and LTC-IC in terms of their overall
light scattering properties.
This was reinforced by additional experiments in which
fraction II wa~ subdivided further into 2-3 additional
fractions. Analysis of these showed that the circulating
CML clonogenic cells were more concentrated in fractions
containing cells with a slightly higher FSC by comparison
to the distribution of LTC-IC in the same fractions. The
high FSC of circulating clonogenic cells in CML patients
differs markedly from the FSC typical of clonogenic cells
in the circulation of normal individuals, but is very
similar to the majority of clonogenic cells in normal BM.
(Sutherland et al., Vol. 74, p. 1563, 1989) Since very
few progenitors were present in fraction III, only cells
~5 in fractions I and II were analyzed in all subsequent
sorts.
Figure 6 shows bivariate contour plots of a single
representative sample of normal (Panels A, C & E) and CML
(Panel B, D & F) light density blood cells in the low SSC
window (fractions I and II in Figure lA). CD34+ cells
(gated as shown by the vertical lines in Panels C and D,
or the horizontal lines in Panels E and F) were subdivided
into CD34+DRLW and CD34+DRhi~h subpopulations as shown by the
horizontal lines in Panel C, or CD34+DRlW, CD34'DR+ and
CD34+DR++ subpopulations as shown by the two horizontal
'` `.: ' : '
.
: .
: ~ :
208025~
- 44 -
lines in Panel D, or CD34~h-123dU~l and CD34+Rh-123bri~ht
populations as shown by the vertical lines in Panels ~ and
F. Unstained cells are shown in Panels A and B.
Figure 6 shows representative distributions of light
density normal and CML blood cells gated for low SSC after
two colour staining for expression of CD34 and HLA-DR, or
expression of CD34 and uptake of Rh-123. A much larger
proportion of light density CML blood cells were found to
express readily detectable levels of CD34 than is the case
for normal blood cells in the same light scatter window
(compare Panels D and F with C and E in Figure 6). The
CML cells also contained a higher proportion of cells that
expressed readily detectable levels of HLA-DR or that
retained Rh-123 by comparison to normal blood. Figures
7 and 8 show the results obtained when the CD34+, SSC~W
cells were sorted according to their expression of HLA-DR
(Figure 7) or Rh-123 uptake (Figure 8) and then analyzed
functionally for clonogenic cells or LTC-IC content.
Figure 7 shows the distribution of clonogenic cells (Panel
A) and LTC-IC (Panel B) within the CD34' fraction of
circulating CML cells subdivided (as shown by the lower
horizontal line in Figure 6D) according to their high or
low expression of HLA-DR ( solid bars). The mean
progenitor recovery + lSEN is expressed as a percent of
the total number light density progenitors recovered
within the low SSC fraction shown in Figure 5A from
studies of 4 different pati.ents. For comparison,
previously obtained analogous results for normal BM (open
bars, n=6) (Sutherland et 21., Vol. 74, p. 1563, 1989) and
normal blood (stippled bars, n=3) progenitors are also
included in this figure.
Figure 8 shows the distribution of clonogenic cells
(Panels A) and LTC-IC (Panel B) within the CD34' fraction
o~ circulating CML cells subdivided (as shown in Figure
. . ~ .~ , .............. .
: . .:, ~
.
: . : . . .
20802~
- 45 -
6F) according to their uptake of Rh-123 (solid bars). The
mean progenitor recovery + lSEM is expressed as a percent
of the total number of light density progenitors recovered
within the low SSC fraction shown in Figure 5A from
Studies of 4 different patients. For comparison
previously obtained analogous results for normal AN (open
bars, n=6 (~6) and normal blood (stippled bars, n=3)
progenitors are also included.
It can be seen that most of ~he clonogenic cells in CML
blood, like most of the clonogenic cells in normal marrow,
expressed readily detectable levels of HLA-DR (Figure 7A)
and showed positive staining with Rh-123 (Figure 8A). In
this respect, however, they both differ markedly from the
clonogenic cells found in normal blood, of which very few
show a DRhi9h or Rh-123bri9ht phenotype. Further subdivision
of the CD34+ DRhi9h fraction of CML blood cells into DR+ and
DR++ subpopulations, as defined in Figure 6D, revealed the
presence of clonogenic cells in both (Tables 3 and 4).
Interestingly, a proportion (~10%) of the clonogenic
progenitors were also found in the DRlW or Rh-123 dUl~
; subpopulations of CD34+ CML blood cells. Although none of
these were specifically genotyped, it is unlikely that
significant numbers in either of these latter
phenotypically defined "normal" subgroups were residual
normal progenitors since normal progenitors, even if
present at normal levels, would have accounted for <10% of
the progenitors in the DRLW (Table 4) or Rh-123
fractions of all patients studied.
When the sorted CML cells were assayed for LTC-IC, the
majority (~75%) were also present amongst the CD34+ DRhi9h
cells (Figure 7B). This is also in contrast to normal
LTC-IC, the majority of which in either blood (~ 100%) or
BM ~ 55%) express little or no HLA-DR. Thus isolation of
CD34+ DRhi9h populations of cells from the peripheral blood
of CNL patients (either DR+ or DR++) yields a highly
. ~ .
.
, ' ' ,
,~ ,.
2080255
- 46 -
enriched LTC-IC population (Table 3). As noted for the
circulating clonogenic cells in the same CML blood
samples, a proportion of the LTC-IC (in this ca~e, ~ 30%3,
was also found in the CD34+DRlW fraction. ~ecause of the
marked elevation in total LTC-IC numbers in these samples,
the number of CD34~DRlw LTC-IC was also consi~tently
greatly ()500x) in excess of values for CD34+DRlW LTC-IC in
the normal circulation (see Table 4~. Similarly, most of
the LTC-IC in the CML blood samples were Rh-123bri9ht (Figure
8B) in contrast to the LTC-IC in either the blood or
marrow of normal individuals. However, on average, ~ 20%
of the circulating LTC-IC in patients with CNL were found
to have a Rh-123dUlL phenotype, of which (1% would have been
anticipated to be residual normal LTC-IC even if these
were still present at normal levels.
It can be seen from Table 2 that the initial frequencies
of the clonogenic cells and LTC-IC in the CML blood
samples studied, although elevated, were quite variable
both on a volume and on a per nucleated cell basis.
Variability was also encountered after these progenitors
were separated into various subpopulations as shown in
Table 3 for light density, CD34 ', DR~W or DRhi~h cells.
However, on average, the purity of circulating CML LTC-IC
in the CD34+DRLW and CD34+DR+ fractions was approximately
10% (Table 2). Corresponding values for the frequency of
clonogenic cells in the CD34+DRLW and CD34+DR+ fractions
were 10~ and 20% (Table 2). As for normal blood, recovery
of LTC-IC in the light density, SSCLW, CD3~+ fraction of
CML blood was high (129~) and of clonogenic cells was
lower (73~) suggesting exclusion of some CML clonogenic
cells with the gating criteria used.
Example 8
Sensitivity of ~MT. progenitors to 4-hydroperoxycyclo-
phosphamide (4-HC)
The present inventors show that LTC-IC in normal blood,
. .
.
2~2~
- 47 _
like LTC-IC in normal BM, are relatively resistant to 4-
HC, as are circulating clonogenic cells, whereas
clonogenic cells in normal BM are more 4-HC-sensitive.
Recent clinical findings indicate that reconstitution of
hematopoiesis with Ph1-negative cells can be achieved in
some CML patients receiving 4-HC-treated autologous BM
transplants. (Carlo-Stella et al., Bone Narrow Transplant,
Vol. 8, p. 265, 1991) This suggests that transplantable
Ph1-positive stem cells may be more sensitive to 4-HC than
normal stem cells. The present inventors have now
evaluated the 4-HC sensitivity of circulating CML
clonogenic cells and LTC-IC and compared these to normal
clonogenic cells and normal LTC-IC. In this series of
experiments, LTC-IC function was assessed in terms of the
clonogenic cell content of LTC evaluated after 4 and 8
weeks (rather than after 5 weeks, as in the studies
described above), since previous experiments, had revealed
differences in the 4-HC sensitivity of normal LTC-IC
measured by these two different endpoints. (Winton et al.,
Exp. Hematol, Vol. 15, p. 710, 1987)
Results for light density CNL blood cells exposed to 100%
~g/ml of 4-HC under standard transplant exposure
conditions (i.e., 2 x 107 cells/ml with 7% red cells for 30
minutes at 37C) are shown in Figure 9, together with
previous data for normal progenitors tested using the same
procedures and reagents.
Figure 9 shows the survival of circulating CML clonogenic
cells (Day O) and LTC-IC (4 and 8 week clonogenic cell
output endpoints) after a brief exposure to 100 ~g/ml 4-HC
(30 minutes at 37C in the presence of 7~ red blood cells,
cells at 2 X 107 cells/ml). Results for circulating CML
progenitors (solid bars showing mean + lSEM for 4
different patients) are shown for comparison together with
previously obtained results for normal BM (open bars, n=6)
and normal blood (stippled bars, n=3) progenitors treated
- '. ' '' ' ~ ' . ~ .
208~2~
- 48 -
using the same conditions.
Circulating CML clonogenic cells and clonogenic cellæ in
normal marrow were simply reduced (to ~ 1~% of initial
numbers) by this treatment. LTC-IC in the same CNL blood
samples appeared only slightly more resistant and were
significantly more sensitive (p~0.01) than normal LTC-IC
from any source.
Example 9
Differentiative potential of ~,MT. LTC-IC
Previous studies have shown that the relative numbers of
different types of clonogenic progenitors present in 5
week-old LTC provides a consistent average overall measure
of the differentiative behaviour of LTC-IC assayed under
standard LTC conditions. (Sutherland et al., Blood Vol.
74, p. 1563, 1989) To assess whether this parameter is
altered in the LTC-IC present in CML blood, the ratio of
BFU-E, CFU-GM and CFU-GEMM numbers before and after LTC of
light density CNL blood cells was assessed. As shown in
Table 4, after 5 weeks in LTC the proportion of
progenitors identified as CFU-GM increased as documented
previously for LTC-IC in the blood and marrow of normal
individuals,
EXAMPLES 10 TO 15
- The materials and methods used in the studies outlined in
~xamples 10 to 15 are detailed below:
Bone marrow cells. Aliquots of normal human marrow cells
were obtained from informed and consenting allogeneic bone
marrow transplant donors at the time of marrow harvests
and with approval of the Clinical Screening Committee for
Research Involving Human Subjects of the University of
British Columbia (Vancouver, Canada). Percolled low-
density cells (~1.068 g/mL) were stained with anti-CD34
antibody directly conjugated to fluorescein isothiocyanate
.~,. , ~ : '
- , . . .
"
2~802~5
- 49 -
(8&12-FITC) (Lansdorp PM et al. J. Exp. Med. 172:363,
1990), and HLA-DR directly conjugated to phycoerythrin
(HLA-DR-PE; Becton Dickinson, Mountain View, CA), and then
~orted on a FACStarP~Us (BD FACS Sy~tems; Becton Dickinson)
(Sutherland HJ, et al. Proc. Natl. Acad. Sci. USA 87:384,
1990). Cells were sorted within low to intermediate
forward light scatter and low 90 light scatter gates to
include cells with properties similar to small
lymphocytes. Cells were additionally sorted for high CD34
expression and very low or negative HLA-DR expression.
This sorting allowed isolation of a subpopulation
representing ~0.4% of total bone marrow cells that was
enriched ~400-fold in cells that produce clonogenic cells
detected after 5 weeks in LTC (Sutherland HJ, et al. Blood
74:1563, 1989; Sutherland HJ et al. Proc. Natl. Acad.
Sci. USA 87:3584, 1990).
Cell line~. ~2 (Mann R, et al. Cell 33:153, 1983) and
NIH-3T3 cell lines were cultured in Dulbecco's modified
Eagle'~ medium (DMEM) with high glucose (4.5g/L) and 10%
heat-inactivated calf serum (for ~2 cells) or 10% fetal
calf serum (FCS). M2-lOB4 cells, a cloned murine (B6C3F1)
marrow fibroblast cell line (Lemoine FM, et al. Exp.
Hematol 16:718, 1988), were maintained in RPMI medium plus
10% FCS. AML-193 cells (Santoli C., et al. ~. Immunol
139:3348, 1987) (American Type Culture Collection [ATCC],
Rockville, MD) were growth in Iscove's medium with 20% FCS
and 10% medium conditioned by the 5637 cell line (ATCC).
NFS-60 cells (Weinstein Y. et al. Proc. Natl. Acad. Sci.
USA 83:5010, 1986), obtained from Dr. J. Ihle (National
Cancer Institute-Frederick Cancer Research Facility,
Frederick, MD), were grown in RPMI with 20~ FCS and 5
pokeweed-stimulated mouse spleen cell conditioned medium.
B9 cells were grown in DMEN with 10% FCS and 100 U/mL IL-6
(Lansdorp PM et al. in Potter M, Melchers F (eds): Current
Topics in Microbiology and Immunology. New York, NY,
Springer-Veriag, 1986, p. lQ5).
- - . . . .
. , -
~' ' " ~ ' ,
- ~ :
20802~
- 50 -
Retroviral vectors and viral producer cell line~.
The retroviral vectors for human GM-CSF and ~-CSF have
been described in Hogge ~E, et al. Blood 77:493, 1991).
Similar principles were used to construct a vector
containing a human IL-3 cDNA. Briefly, an IL-3 cDNA ( from
Genetics Institute, Boston, MA), was truncated at the 3'
end to remove A-T rich sequences thought to be responsible
for destabilizing mRNA transcripts (Shaw G., Kamen R. Cell
46:659, 1985). This 632-bp cDNA frag~ent was linked to a
250-bp PvuII-BglII fragment from the pXl vector containing
the promoter from the herpes simplex thymidine kinase (tk)
gene (Anderson WF. et al. Proc. Natl. Acad. Sci. USA
77:5399, 1980) and the tk-IL-3 cassette inserted into the
Xho 1 site in the N2 retroviral vector, which is 3' of the
neor gene (Eglitis MA, et al. Science 230:1395, 1985).
Retroviral constructs were transfected into the c2
ecotropic packaging cell line (Mann R., et al. Cell
33:153, 1983). Individual clones of G418 resistant (G418r)
transfected cells were isolated, expanded, and assessed
both for viral titer by the ability of their growth medium
to generate G418r NIH-3T3 cells, and for the production of
growth factor bioactivity on growth factor-responsive cell
lines. Clones producing viral titers greater than 105
colony-forming units/mL were used to infect N2-lOB4 cells.
Stromal feeders. Irradiated (15 Gy of 250-kV peak x-rays)
normal human marrow adherent layer feeders (NF) were
prepared as previously described (Sutherland HJ, et al.
Blood 74:1563, 1989; Sutherland JH, et al.Proc. Natl.
Acad. Sci. USA 87:3584, 1990). To generate human growth
factor-producing M2-lOB4 feeders, cell-free growth medium
was harvested from viral producer cells and, together with
8~g/mL polybrene, added to subconfluent cultures of N210-
B4 cells. After 4 hours of incubation at 37C the virus-
containing medium was replaced with standard growth
medium. Forty-eight hours post-infection the cells were
typsinized, replated in growth medium containing 0.4 mg/mL
...... . . . .
., . - ~ :
..
20802~
- 51 -
G418, and the cells grown to confluence, at which time the
growth medium was tested for growth factor bioactivity.
Mass cultures of these retrovirally infected, G418r, growth
factor-producing M2-lOB4 cells were subsequently
mzintained and passaged as continuous cell line~. Using
standard techni~ues and hybridization of blots to 3ZP-
oligolabeled GM-CSF, G-CSF, or IL-3 cDNA probes, Southern
and Northern analysis (Feinberg AP, Vogel~tein B Anal.
Biochem. 132:6, 1983; Sambrook J., et al.: Nolecular
cloning: A laboratory manual. Cold Spring Harbor, NY,
Cold Spring Harbor Laboratory, 1989) showed grossly intact
proviral DNA and the expected full-length and spliced
retroviral transcripts in the infected M2-lOB4 cells. N2-
lOB4 feeders were prepared before the initiation of
cocultures by seeding 3 x 105 M2-lOB4 cells into 35-mm
corning tissue culture dishes (Corning Glassworks, Corning
NY) or into Nunc 96-well plates (A/S Nunc, Roskilde,
Denmark) at 104 cells per well. In cultures containing
cells from more than one growth factor-producing cell line
(to test the effect of specific combinations of growth
factors), equal numbers of each of the types of cells were
used, keeping the total cells plated constant at the
values given above. All M2-lOB4 feeders were irradiated
with 80 Gy of x-rays.
Coculture~. In a total of 16 experiments, 800 to 11,000
sorted human bone marrow cells were placed in cultureR
with or without feeders (as indicated) in LTC medium.
Cultures were then maintained at 33C for 5 weeks with
weekly half-medium changes as previously described
(Sutherland HJ, et al.: Blood 74:1563, 1989; Sutherland
JH, et al. Proc. Natl. Acad. Sci. USA 87:3584, 1990). At
the end of 5 weeks, all nonadherent cells were removed and
counted, and the adherent cells were then suspended by
trypsinization. Alquots equal to 1/3 to 1/2 of total
adherent and nonadherent cells were plated in standard
methylcellulose cultures for assessment of total
,
2080255
- 52 -
erythropoietic (BFU-E), granulopoietic (CFU-GM), and
multilineage (CFU-GEMM) progenitors detected 20 days after
initia~ion (Cashman JD., e~ al. Blood 75:~6, 1990).
Aliquots were also reseeded on top of new irradiated
normal human NF in 96-well plates for assessment of LTC-IC
content by limiting dilution analysis and measurement of
total clonogenic cell content after an additional 5 weeks
(Sutherland JH, et al. Proc. Natl. Acad. Sci. USA
87:3584, 1990).
Growth factor bioactivity. Growth factor bioactivity was
measured in growth media collected 2 days after a complete
change of the medium in confluent cultures of viral
producer cells or feeders, and in media removed from
cocultures at weekly intervals. Bioactivity was measured
by comparing the stimulation of 3H-thymidine incorporation
into appropriate growth factor-responsive cell lines to
that obtained with recombinant growth factor standards.
Recombinant GN-CSF and IL-3 were gifts from Biogen
(Geneva, Switzerland) and Behring (Frankfurt, Germany),
and recombinant IL-6 was purchased from R & D Systems,
Inc. (Minneapolis, MN). Recombinant G-CSF was purchased
from Amersham (Oakville, Canada). GM-CSF and IL-3 levels
were measured on human ANL 193 cells, G-CSF on NFS 60
cells, and IL-6 on B9 cells.
Example lO
Growth factor production by engineered M2-lOB4 cells.
Human growth factor-producing N2-lOB4 cells were generated
by infection of the cells with ecotropic retrovirus
capable of the transfer and expression of both the neor
gene, which renders eukaryotic cells resistant to the
neomycin analogue G418, and the cDNAs for either human GM-
CSF, G-CSF, or IL-3. When retrovirally infected M2-lOB4
cells had grown to confluence under G418 selection,
samples of their growth medium were tested for growth
factor bioactivity (Table 13. Bioactivity was detected
':' : . - ,
' ~ : ' ' . , , '
20802~5
- 53 -
only from cells infected with the appropriate virus and
the levels measured ranged from ~l to 20 ng/mL.
BioactiYity from cultures containing two or three types of
growth factor secreting M2-lOB4 cells was twofold to
threefold lower, consistent with the lower number of each
type of cell in these cultures. These remained stable for
at least 2 months in the absence of G418 selection, even
after the cells were irradiated. Bioactivity at levels
approximately e~ual to the levels from the feeders alone
; 10 was also detected in media removed from cocultures of M2-
lOB4 cells with purified human marrow cells, and levels
remained unchanged throughout the period of the
experiments. In cocultures with uninfected M2-lOB4
feeders no bioactivity could be detected. Assays for IL-6
were also performed on media conditioned by M2-lOB4 cells
and media removed weekly from cocultures. These assays
showed IL-6 levels to be consistently less than 0.01
ng/mL. In previous experiments the concentration of
purified recombinant growth factor required to stimulate
half-maximal hematopoietic colony growth from nonadherent
marrow cells placed in short-term methylcellulose assays
has been shown to be 0.01 ng/mL for GM-CSF, 10 ng/mL for
G-CSF, and l ng/mL for IL-3 (Hogge DE., et al. Blood
77:493, l991), suggesting that growth factor production by
the retrovirally infected M2-lOB4 cells was sufficient to
warrant testing these cells as feeders in LTC.
xample 11
Capacity of N2-lOB4 cells to support human hematopoiesis.
Total numbers of nonadherent cells, clonogenic cells, and
LTC-IC in cocultures 5 weeks old were measured to assess
the ability of control (uninfected and/or N2-infected) M2-
lOB4 cells to support hematopoiesis at these three levels
of hematopoietic cell development (Table 2). Results
obtained in each case were compared with those obtained
from cultures containing normal human MF or no feeders
2~8~255
- 54 -
(i.e., hematopoietic cells seeded directly onto plastic).
Despite the lack of detectable G-CSF, GM-CSF, IL-3 or IL-6
in cultures containing control M2-lOB4 cells,
significantly support for all levels of hematopoiesis was
evident by comparison to results for cultures without
feeders. For LTC-IC maintenance and production of
clonogenic cells, M2-lOB4 cells were almost as effective
as normal human MF. However, human MF did offer a
significant improvement over M2-lOB4 cells when effects on
terminal cell numbers (nonadherent cell production) were
assessed.
Example 12
Specific growth factor effects on terminal hematopoiesis.
Nonadherent cell numbers in cultures 5 weeks old
containing growth factor-producing M2-lOB4 cells were
compared in a paired t-test to cultures with control M2-
lOB4 cells (Fig. 1) and to cultures with human NF. IL-3-
producing M2-lOB4 cells alone were not different than
control N2-lOB4 cells (P = .4~. However, all other types
of growth factor-producing feeders, either alone or in
combination, increased nonadherent cell output above that
seen with control M2-lOB4 feeders (P ~ .005). GM-CSF-
producing M2-lOB4 cells with or without other growth
factor-producing M2-lOB4 cells were most effective in this
regard. Alone, they supported the production of ~20 times
more nonadherent cells than cultures containing control
M2-lOB4 cells, and N4 times more nonadherent cells even
than cultures containing human MF (P ~ .005). Although
the combination of IL-3- and G-CSF-producing M2-lOB4 cells
was less effective for the promotion of terminal cell
amplification than any feeder producing GM-CSF, it was
equivalent to human MF in this regard.
The maintenance of LTC-IC was found to be supported by
control murine stromal cells as effectively as by standard
' , "'
.~: . . :
.. . . ..
- ~ .- :~ . ,
:-: : : -
-
,. .. .
20802~
human marrow adherent layers. The presence of G-CSF and
interleukin-3-producing M2-lOB4 cells in combination was
found to further enhance the maintenance and early
differentiation of these cells without a decline in their
proliferative potential as measured by the clonogenic
output per LTC-IC. However, this effect was lost if GM-
CSF-producing feeders were also present. On the other
hand, in the presence of GM-CSF-producing feeders, the
output of mature granulocytes and macrophages increased
20-fold.
Example 13
Specific growth factor effects on clonogenic cell output.
Production of clonosenic cells was analyzed in the same
experiments by comparison of numbers of clonogenic cells
in 5-week-old cocultures to contxols using a paired t-test
(Fig. 2). G-CSF feeders alone and G-CSF plus IL-3-
producing feeders provided more support than control M2-
lOB4 feeders (P ~ .05) and G-CSF plus IL-3 feeders were
twice as supportive as human NF, although this later
differ~nce did not quite reach statistical significance (P
= .12). GN-CSF feeders either alone or in combination
with G-CSF or IL-3 feeders resulted in clonogenic cell
output values at 5 weeks that were close to those obtained
in cultures with control M2-lOB4 cells. To distinguish
whether the IL-3-plus G-CSF-producing feeder combination
increases clonogenic cells by increasing the number of
LTC-IC recruited to differentiate, or by increasing the
proliferative ability displayed by the LTC-IC originally
present, an additional series of experiments was
undertaken. In these experiments, sorted normal bone
marrow cells were seeded at a limiting dilution onto
either IL-3-plus G.-CSF- producing M2-lOB4 cells or human
MF, and the frequency and avexage clonogenic cell output
by individual LTC-IC was then determined from a knowledge
of the clonogenic content of wells measured 5 weeks later.
, '~ '
208025~
- 56 -
The results from five such experiments suggest a slight
but not statistically significant advantage for the IL-3-
plus G-CSF-secreting M2-lOB4 cell~ for both parameters
assessed, i.e., the proportion of initially seeded cells
detected as LTC-IC was 1.4% and 1.1% and the averag~
number of clonogenic cells produced per LTC-IC detected
was 5.5 and 4.6 for the IL-3 + G-CSF feeders and human NF,
respectively.
Example 14
Specific growth factor effects on LTC-IC maintenance. By
plating cells at limiting dilution on human NF, the
absolute number of LTC-IC in a population can be
quantitated (Sutherland JH, et al. Proc. Natl. Acad. Sci.
USA 87:3584, 1990). This analysis can be performed on
cells removed at various time points from a culture to
provide a measure of the ability of the conditions
prevailing in the cultures to promote the maintenance
and/or self-renewal of LTC-IC. Approximately 25% of the
` number of input LTC-IC were detected after 5 weeks in LTC
initiated by seeding sorted marrow onto human NF. (Mean
+ SEM LTC-IC per 1,000 sorted cells originally plated =
16.7 + 4.0 on day O, and = 4.3 + 1.0 at 5 weeks, in six
experiments). The ability of control and growth factor-
producing N2-lOB4 cells to maintain LTC-IC was similarly
assessed by quantitating the number of LTC-IC remaining
after 5 weeks in primary cultures containing various
feeders. As shown in Fig. 3, the combination of IL-3 plus
G-CSF feeders in the primary cultures allowed better
maintenance of LTC-IC than control M2-lOB4 feeders (P <
.05) and was even somewhat better than human NF.GM-CSF-
producing feeders alone, or together with G-CSF-producing
feeders, provided less LTC-IC maintenance than human MF (P
.05) and any culture that contained GM-CSF-producing
feeders appeared worse than control N2-lOB4 cells for LTC-
IC maintenance (P = .14 to .18). The other feeder
combinations tested provided support of LTC-IC maintenance
'., ' '`, . :'
:: '
208025~
that did not differ significantly from that obtained with
human MF or M2-lOB4 cells.
Example l~
Lack of any growth factor effect on the proliferative
potential displayed by LTC-IC present after 5 weeks in
culture.
In addition to determining the number of LTC-IC maintained
under various coculture conditions, the proliferative
potential of these cells, as indicated by the average
number of clonogenic progenitors produced per LTC-IC
(CFU/LTC-IC) before and after culture, was measured by
limiting dilution analysis. CFU/LTC-IC was the same for
LTC-IC maintained on human MF for 5 weeks as for the LTC-
IC in the original purified marrow sample (4.0 + 0.7 v 4.3
+ 0.4). Moreover, despite the fact that the number of
LTC-IC maintained in primary cocultures with various types
of growth-factor producing M2-lOB4 cells varied from
twofold higher to threefold lower than the number of LTC-
IC maintained in cultures containing human MF, the
proliferative potential of the Lrrc-Ic present after 5
weeks was not influenced by the type of feeder used in the
primary culture (analysis of variance, P = .46) (Table 3~.
EXAMPLES 16 TO 19
The materials and methods used in the studies outlined in
Examples 16 to 19 are detailed below:
CELLS peripheral blood mononuclear cells were obtained
with informed consent from normal volunteer donors as a
byproduct of plateletphereses performed at the Vancouver
General Hospital, Canada. Cells were further depleted of
T cells by incubation with 2-aminoethylbromide
isothiouronium-treated sheep blood cells for 30 minutes at
4C and subsequent isolation of the light density
(1.077gm/cm3) fraction after centrifugation on Ficoll-
.:
::
. ~, :
208025~
- 58 -
hypaque (FH) as described previously. (Marsden M. et al,
J. Immunol Methods, 33:323, 1990.) Random checking of this
procedure showed that less than 2~ of the recovered cells
were CD2 positive (T cells) by FACScan analysis. Normal
~one marrow (BM) aspirate cells ere obtained with informed
consent from normal donors of allogenic morrow for
transplantation. BM cells were either used directly, or
after lysis of contaminating red blood cells by brief
exposure to ammonium chloride, (Turhan AG et al, N. Engl.
- 10 J. Med. 320:1655, 1989) or after centrifugation on FH as
indicated.
CULTURES. Cells from primary blood or BM samples or from
LTC were assayed for erythroid (BFU-E), granulopoietic
(CFU-~M~, and multilineage (CFU-GEMM) colony-forming cells
in standard methylcellulose cultures containing 3 units
per ml of human erythropoietin and 10% agar-stimulated
human peripheral leukocyte conditioned medium. This
methodology and the criteria used for colony recognition
have been described in detail in Cashman J. et al, Blood
66:1002, 1985. LTC-IC assays were initiated by seeding an
aliquot of the test cell suspension into cultures
containing irradiated (1500 c~y) allogenic marrow cells (3
X 104 per cm2) that had been subcultured from the adherent
layer of previously established 2-4 week old LTC.
(Sutherland HJ et al, Blood 74:1563, 1989; Eaves CJ et al,
J. Tissue Culture Methods 13:55, 1991.) LTC-IC assay
cultures were then fed weekly by replacement of half of
the growth medium containing half of the nonadherent cells
with fresh growth medium (-medium supplemented with
inositol, folic acid, glutamine, 10-4 M 2-mercaptoethanol,
10-6 M hydrocortisone sodium hemisuccinate, 12.5% horse
serum and 12.5 fetal calf serum (FCS). In most
experiments LTC-IC assays were performed in cultures set
up in 2.5 ml. volumes in 35 mm tissue cultures dishes,
although for the limiting dilution assays, smaller,
appropriately scaled down (0.1 ml) cultures were used as
~:
-
20802~S
- 59 -
described previously. (Sutherland HJ, Proc. Natl. Acad.
Sci. USA 87:3584, 1990.) After a total of 5 weeks (unless
specified otherwise), the nonadherent cells were removed,
washed and combined with cells harvested from the adherent
fraction by trypsiniazation. (Lansdorp PM et al, J. Exp.
Med. 172:363, l990.) These cells were then adjusted to a
concentration suitable for plating in methylcellulose
assays (to yield <200 colonies per 1.1 ml assay culture.
For a detailed description of the LTC-IC assay procedure,
see Eaves CJ et al, J. Tissue Culture Nethods 13:55, 1991.
In the experiments reported there (unless specified
otherwise), the number of clonogenic cells present in LTC
harvested after 5 weeks (i.e. the number of BFU-E plus
CFU-GM plus CFU-GENM present in both the nonadherent and
adherent fractions at this time) was used to provide a
quantitative, albeit relative, measure of the number of
LTC-IC originally seeded into the LTC. However, this
number of clonogenic cells can be directly converted to an
absolute number of LTC-IC simply by dividing by 4, since
this is the average number of clonogenic cells calculated
to be present in 5 week-old cultures per initial LTC-IC
seeded.
STAINING AND FLOW CYTONETRY. Cells were prepared for
staining by resuspension in Hank's solution containing 2~
FCS and 0.01~ sodium azide (HFN). They were then
incubated with 1-2 ~g/ml of anti-HLA-DR-phycoerythrin (PE)
(107 cells/ml) or RH-123 at a final concentration of 0.1
~g/ml as described in Sutherland HJ et al, Blood 74:1563,
1989, and Udomsakdi C. et al, Exp. Hematol 19:338, 1991.
In some cases cells were stained with an anti-CD34
antibody (8Gl2)36 directed conjugated to PE or fluorescein
isothiocyanate (FITC). Stained cells were scored using a
Becton Dickinson FACStar Plus (FACS) equipped with an
argon laser emitting at 488 nm. Fluorescence of RH-123,
FITC-, and PE-labelled cells was measured using 530/30 and
575/?6 band pass filters, respecti~ely, after calibration
: .:
:
2~802~5
- 6~ -
of the FACS prior to each sort using 10 ~m fluorescent
beads. In some experiments, cells were gated according to
their forward light sca~ter characteristics (FSC) and side
scatter characteristics (SSC) to exclude most erythrocytes
and granulocytes, as described in Sutherland HJ et al,
Blood 74:1563, 1989, and Udomsakdi C. et al, Exp. Hematol
19:338, 1991. Cells appearing in this light scatter
window (see Figure 4A, fractions I and II) constituted
>60~ of the total density fraction T cell depleted blood
cells. Cells were collected after sorting in Hank's
solution containing 50~ FCS and were maintained at 4C
until plated.
~xample 16
QUANTITATION OF LTC--IC IN NORMAL BLOOD.
In an series of experiments, the number of clonogenic
cells present after 5 weeks in LTC initiated with T cell-
depleted suspensions of normal peripheral blood
mononuclear cells seeded onto pre-established, irradiated
marrow adherent layers was found to be a linear function
of the number of cells initially added over a 1000-fold
range of input cell numbers. Results for a representative
experiment are shown in Figure 13.
Figure 13 shows a linear relationship between the number
of light density (~1.077g/cm3) T cell-depleted peripheral
blood cells from a representative normal individual seeded
onto pre-established, irradiated normal marrow feeders and
the total number of clonogenic cells detected when these
LTC were harvested and assayed in methylcellulose 5 weeks
later. The slope of the regression line fitted to this
data set is 0.92 + 0.09.
Three such dose response experiments also included a
series of assay cultures (20-25 per point) which were
seeded with limiting numbers of LTC-IC (i.e. ~1 LTC-IC per
assay culture). From the proportion of positive and
.. . ~ ; . ~ . . : .
- ... ' . . ~ . .
.
- ~ ' -:
, . . . ..... - ~ : . ...
- : -
2~80255
- 61 -
negative assay cultures (containing >1 clonogenic cell
each, or none, respectively,) absolute frequencies of LTC-
IC in the original test cell suspension were calculated
using Poisson statistics (Porter EH et al, Br. J. Cancer
17:583, 1963, and Taswell C., J. Immunol 126:1614, 1981)
(Figure 14). Figure 14 shows the limited dilution
analysis o data from a representative experiment in which
decreasing numbers of light density T cell-depl~ted normal
peripheral blood cells were seeded onto irradiated marrow
feeders and the number of clonogenic cells detectable
after 5 weeks was then determined. For this experiment,
the frequency of LTC-IC in the starting cell suspension
~i.e., the reciprocal of the concentration of test cells
that gave 37% negative cultures) was 1 per 1.5 x 105 cells
(95% confidence limits = 1 per 9.9 x 104 - 1 per 2.2 x 105
cells). From this value and a knowledge of the total
number of clonogenic cells produced by a large number of
cells of the same input suspension, the avera~e 5 week
output of clonogenic cells per LTC-IC in normal blood was
calculated. This value was found to be ~.7 + 1.2 (see
Example 5) which is similar to the value of 4.3 + 0.4 that
we reported for LTC-IC in normal BM. (Sutherland HJ, Proc.
Natl. Acad. Sci. USA 87:3584, 1990.) Bulk measurements of
the 5 week clonogenic cell content of assay cultures
initiated with T cell- depleted blood samples from other
normal adults could then also be used to derive absolute
LTC-IC per ml values using this average clonogenic output
per LTC-IC conversion factor. Table 9 shows the average
concentration of LTC-IC in the peripheral blood calculated
from values measured on 23 normal adults, together with
the average concentration of circulating clonogenic cells
(BFU-E plus CFU-GM plus CFU-GEMM) obtained for the same 23
samples. The derived value of ~3 LTC-IC per ml is ~75-
fold lower than the concentration of circulating
clonogenic cells both measured here (Table 9) and reported
previously. (Sutherland HJ. et al, A Practical Guide. Boca
Raton, CRC Press Inc., 1991, pp 155, and Ogawa M. et al,
~ . . .
.
20802~5
- 62 -
Blood S0:1081, 1977.) Hence the frequency of LTC-IC
relative to other nucleated cells in the blood (~l per 2
X 106) is ~100-fold lower than the frequency of LTC-IC
relative to other cells in the BM. (Sutherland HJ, Proc.
Natl. Acad. Sci. USA 87:3584, 1990.)
Example 17
PHENOTYPE OF CIRCULATING LTC-IC.
The distribution of LTC-IC and clonogenic cells in various
phenotypically-defined subpopulations of the T cell-
depleted, light density fraction of normal peripheral
blood were then assessed. ~hese were obtained using the
FACS to separate cells on the basis of their light
scattering properties, expression of CD34, HLA-DR, and RH-
123 uptake.
Figure 15 shows the bivariate contour histograms of light
density T cell-depleted normal peripheral blood cells
stained with anti-CD34 and anti-HLA-DR. Panel B shows the
distribution of these cells in the low side scatter window
(fraction I ~ II in Figure 4A). Panel C shows the
distribution of HLA-DRhi9h and HLA-D~W cells after also
gating for CD34~ cells as indicated in Panel B. The light
scattering properties of CD34'HLA-DRLW and CD34~ELA-DRhi9h
cells are demonstrated in Panels E and F, respectively.
Unsorted, unstained control and irrelevant (lD3) antibody-
stained cells are shown in Panels D and A, respectively.
As illustrated in Figure 15, even after removal of the R
cells from the light density fraction of leukapheresis
samples, the frequency of cells expressing readily
detectable levels of CD34 (as defined by the vertical gate
shown in Figure 15B) was still very low (0.1-0.5%) as
compared to the non-T cell-depleted light density fraction
of normal marrow, where values of 1-4% are typically
obtained, (Civin CI. et al in Knapp W. et al (eds):
Leucocyte Typing IV. White Cell Differentiation Antigens,
Oxford, Oxford University Press, 1989, pp 818) even using
- ~ . , ........... . ~ . . .
.
20802~5
- 63 -
~imilarly stringent gating criteria. (Sutherland HJ et al,
Blood 74:1563, 1989.~ Most of the cells in the fraction
defined as CD34~ expressed no or low levels of HLA-DR
(Figure 15C), and had low SSC properties (Figure 15E and
F). Cells with a CD34~ and HLA-DRlW phenotype (defined by
the horizontal gate shown in Figure 3C) were found almost
exclusively amongst the smallest light density cells (low
FSC, Figure 15E).
Figure 16 shows the light scatter profiles of T cell-
depleted light density normal blood cells (Panel A). The
mean ~ SEM of the percentages of nucleated cells (open
bar), clonogenic cells (stippled bar), and LTC-IC (solid
bar) in each sorted fraction are shown in Panel B (n=4).
Figure 16 shows the distribution of LTC-IC and clonogenic
cells observed when the total light density T cell-
depleted fraction of peripheral blood cells was subdivided
into 3 populations defined by their light scattering
properties: l-low FSC, intermediate SSC; II - intermediate
to high FSC, low SSC; and III - all remaining cells (i.e.
open FSC, intermediate SSC). Although each gated
population contained approximately equal number of cells,
virtually all LTC-IC and most of the clonogenic cells were
consistently found in the fraction containing the smallest
cells (I). No LTC-IC and less than 5% of all clonogenic
cells were found in fraction III. Therefore subsequent
sorts, only cells in the low SSC fractions (I and II) were
analyzed.
Figure 17 shows a representative histogram of CD34~, light
density T cell-depleted normal blood cells (in the
previously described low SSC window shown in Figure 16A)
double-stained with PE conjugated anti-HLR-DR. CD34+HLA-
DRL~ cells were further subdivided into CD34~HLA-DR
(fraction 1) and CD34+HLA-DR~ (fraction 2) cells as shown
in Panel A. The remaining cells are CD34'HLA-DRhi9h,
: .
~ ,
.
,
208025~
- 64 -
indicated as fraction 3 in Panel A and referred to as
CD34'HLA-DR~ cells in Panel B. The dark histogram in Panel
A shows the profile for unstained cells. This mean + SEM
of the percentages of nucleated cells (open bar),
clonogenic cells (stippled bar), and LTC-IC (solid bar) in
each sorted fraction are shown in Panel B (n=3).
Figure 17 shows the results of functional assays performed
on cells sorted both according to their expression of CD34
and HLA-DR. In this case, only CD34' cells were assayed.
These were then divided into HLA-DRhi~h and HLA-DR~W cells
were further subdivided into an HLA-DR- and an HLA-DR~
population. No LTC-IC and very few directly clonogenic
cells were detected in the CD34~HLA-DRhi3h fraction.
However, further subdivision of the remaining CD34~, HLA-
DRLWcells did allow some differential separation of LTC-IC
and directly clonogenic cells, moxe of the latter (~40% vs
~10% LTC-IC) being found in the HLA-DR~ fraction. Table
10 shows the enrichment and recovery values obtained for
LTC-IC and clonogenic cells in various HLA-DR
subpopulations of the light scatter gated, CD34' fraction,
as compared to the unstained, light density, T cell-
depleted starting population in these experiments.
Recovery of LTC-IC in the CD34', HLA-DRLW fraction was
>100% in all 5 experiments performed suggesting that all
circulating LTC-IC express readily detectable levels of
CD34, as do those in normal BM. (Sutherland HJ et al,
Blood 74:1563, 1989.) Isolation of a rare subpopulation
of circulating cells defined by the same properties as
previously used to purify BM LTC-IC (i.e. low density, low
forward light scatter, high expression of CD34 and low
expression of HLA-DR), allowed a much greater enrichment
(>l,000-fold beyond the light density, T cell depletion
step) of circulating LTC-IC to be routinely obtained.
Thus, even though the initial frequency of LTC-IC in
normal peripheral blood as much lower (on a per cell
basis), the final purity of LTC-IC achievable from normal
- - : . ,
208025~
- 65 -
peripheral blood using these parameter~ (~0.5-1%, Table
10~ was approximately the same as the best yet described
for normal BM. (Sutherland HJ, Proc. Natl. Acad. Sci. USA
87:3584, 1990.) Recovery of clonogenic cells in these same
experiments appear to be somewhat lower (Table 10),
suggesting that some circulating clonogenic cells may have
been excluded from the CD34~ population gated for in these
studies, or that subopitimal plating efficiency of
clonogenic cells may have been achieved when highly
purified populations were assay. Failure to detect
additional clonogenic cells in the higher FSC/SSC
fractions (II and III, Figure 16) due to potential
inhibition of their colony-forming ability by the presence
of increased numbers of monocytes were ruled out by mixing
experiments (i.e. no reduction of clonogenic cells
detected when cells from Fraction I were mixed with cells
from Fraction III in a 1:2 ratio).
The results of combined staining for CD34 expression and
RH-123 uptake are shown in Figure 18. In particular,
Figure 18A is a representative histogram of CD34', light
density T cell-deplet0d normal blood cells double-stained
with Rh-123 and sorted into CD34+RH-123dULL and CD34'RH-
123bri9ht fractions (fractions 1 and 2, respectivel~; Panel
A). The dark histogram in Panel A shows the profile for
unstained cells. The mean + SEM of the percentages of
nucleated cells (open bar), clonogenic cells (stippled
bar), and LTC-IC (solid bar) in each sorted fraction are
shown in Panel B (n-3).
In this case, no difference was noted between circulating
LTC-IC and clonogenic cells in terms of their distribution
between the CD34' RH-123bri9ht fractions with mor than 80% of
both being found in the Rh-123dUlL but most of the
clonogenic cells are Rh-123br;9ht, thus allowing their
differential isolation by sorting according to this
parameter. (Udomsakdi C. et al, Exp. Hematol 19:338,
., ~
. .
,.
.
2080255
- 66 -
1991.) Nevertheless, the final purity of LTC-IC in the
light density, T cell-depleted, CD34', Rh-123dUl~ fraction
of normal blood was similar to that obtained by ~electing
for HLA-DRIw cells (Table 10) or by application of the same
criteria to BM. (Udomsakdi C. et al, Exp. Hematol 19:338,
1991.) This reflects a similarly greater overall
enrichment achieved with blood versus mArrow using either
HLA-DE expression or retention of Rh-123 as the final
separation parameter.
Example 18
4-HC SENSITIVITIES OF CIRCULATING PROGENITORS. Because
normal circulating clonogenic cells were known to be a
~uiescent population (Eaves CJ et al in Goldman JM (ed):
Bailliere's Clinical Haematology. Vol. 1, #4. Chronic
1~ Myeloid Leukaemia, London, sailliere Tindall, 1987, pp
931) and appeared phenotypically to be more similar to
LTC-IC in either blood or BM than to the clonogenic cells
found in the BM, it was of interest to compare the
sensitivities of circulating clonogenic cells and LTC-IC
to 4-HC, using the same type of treatment protocol that is
in widespread clinical use for treating autologous marrow
transplant. In this set of experiments, LTC-IC function
(~efore or after exposure to 4-HC) was assessed in terms
of the clonogenic cell content of assay cultures evaluated
after 4 and 8 weeks (rather than after 5 weeks as in the
experiments described above), since previous experiments
and revealed differences in LTC assays of BM samples for
autologous transplant when these two time points were
compared. (Winton EF et al, Exp. Hematol 15:710, 1987, and
3Q Eaves CJ et al, Blood Cells (in press).) Results for LTC-
IC and clonogenic cells in normal peripheral blood and BM
are shown in Figure 19.
Figure 19 shows a comparison of the number of clonogenic
cells and LTC-IC surviving a 30 minute exposure to
lOO~g/ml of 4-HC at 37C with 7% erythrocytes present.
Values shown are the mean + SEN of the percentages of
.
- ' -~
:~ ' ' ,
2080255
- 67 -
clonogenic cells and LTC-IC from normal BM (open bars) and
normal blood (solid bars) as a percent of values for
control cells (n=3 or BM and n=4 for blood cells).
A dramatic difference in the effect of a 30 minute
exposure at 37C to lOO~g/ml of 4-HC on the viability of
clonogenic cells and LTC~IC appear to be similar to BM
LTC-IC in their relative resistance to 4-HC. For LTC-IC
from both sources, a slight increase in 4-HC resistance
was noted for LTC-IC defined by the longer clonogenic cell
output endpoint (i.e., 8 weeks).
'
Example 1~
DIFFERENTIATIV~ POTENTIAL EXPRESSED BY CIRCULATING LTC-IC
IN LTC. Table 11 shows the relative proportions of BFU-E,
CFU-GM and CFU-GEMM in the total clonogenic population of
5 week-old LTC initiated with circulating LTC-IC of
varying purities,and compares these to the relative
numbers of these same types of clonogenic cells in the
original blood samples. Data for unseparated and LTC-IC
enriched cell populations from normal BM obtained in
previous studies (Sutherland HJ et al, Blood 74:1563,
1989) are also shown in Table 11 for comparison. It can
be seen that the differentiative behaviour exhibited by
LTC-IC in normal blood and BM is similar and is also not
affected by the purity of the ~TC-IC in the starting
population. In both cases, a significant skewing towards
the generation of CFU-GM by comparison to the number of
CFU-GM and BFU-E actually found in normal blood or BM was
observed. To some extent this might be expected because
all stages of granulopoietic cell differentiation are
supported in the LTC system whereas erythropoiesis appears
to be blocked at the stage of mature BFU-E production.
(Coulombel L. et al, Blood 62:291, 1983.) As a result,
this latter contribution to total BFU-E numbers in vivo is
absent from LTC-derived populations.
- ,
~ ~ .
' :" -: . -- -
208025~
- 68 -
The present invention has been described in detail and
with particulsr reference to the preferred embodiments;
however, it will be understood hy one having ordinary
skill in the art that changes can be made thereto without
departing from the spirit and scope thereof.
J~. . ~ ' ' , . ' ' ' . '
,~'',, , ~',.' ' ~' ' '' ` '
. ~ . ' . '' , '
' ' ' . ' ' ' . ' .
.' ' .' ' ' ' :- '
, " ' :
-:: . ', ' :
' .; ~ ' '
- 69 - 20802~5
TAE~LE 1
No. of dono~e~c cdl~ per LTC-IC
~5wk~
Normal BM 4.3 t 0.4 (5~
Nomlal Blood 3.7 ~ 1.2 (3)
CML Blood 3.1 + 0.416)
Mean +SEM of lralue~ from ~3~) experlmc~t~ calculatcd by multlplylng the
frequcncy of LTC-IC In cach acperiment (dcterm~ed by llmltlng dllutlon
assays) by the total number o~ celb plated In dl LTC to determlne the total
number of LTC-IC for that exper~erlt. The total content of clonogenlc
progenlto~s In all LTC for ~n ~Idual ~ent was obtained dlrectly grom
clonogen1c progenltor ass~rs.
- . ,
~ 70 - 20802~5
TABLE 2
Frequency of Pr~mlUvc Progenltors In the CML PaUcnts 8tudled
Patlcnt No. WBC/ALClono8cnlc Ccll~/ml, LTC-lC/mLa
Ix 10~
.
2g.000 17,000
2 156 704,0G0 266,000
3 137 .72.000 1~,000
4 62 82,000 7.200
262 1.060,000145,000
6 104 161,u~10 8.300
7 1 10 86,000 1.300
8 142 344,000 12,500
9 ~36 1,090,000 10,000
Mean + SEM162 ~ 40403,000 + 145,00052.000 + 31,000
a These numbers rcprcsent absolute LTC-IC valucs calcubted ~s descrlbed h the Mdhods
.. . .. .
. - .
- : ~:
:. .'
-- 71 --
20802~5
~ ~ 5 e 8
~ ~Y~. ;;~
c y ; ~ 5 ~ ¦ ~ J ~ 5
:~ ~ . ~ ~ ~ i ~; 5 ~
5 ~ a ~ ~ 9 ~ e
t I i 3 ~ 5 ` e i
~ t~ 1 ~ ~ ~ ~ C
3 ~ ~ ~ i i l ~ ia~ ~ ~
a a a ,,,,," " " v_
, , " ., ; -
:
-- 72 --
2080~55
a 5 "
~Z ~ 0~0~ j COCO~O~ CO ~0~ j . R' ~
R5 ~ 888 88 O 80 ~ ~ ~ 3 ~ 5,
R~ ~ ¦ ~b--~ - 0 j ~IR a3
~U ~ ~ ~3~5 ~ R~5 ~ f 5
R R ~ ~ ~ -O N ~o ~N~ R !~ ~ ~
~ 8 ~ N ~ ~ ~ ;N r~ 5 ~ f ~ D D
~ ~ ~ ~ e ~ g ~ z
.
20802~
TABLE 5
Relattve Numbers IExpressed as a Pcrcent of the Total) of Dlfrerent l~pcs o~
Clonogcnlc CeUs Prescnt In CML E~lood and Produccd by LTC-IC In CML Blood a~er 5Weelcs In LTC
O~lgln o~ No. d Clonogenlc CcL~ l,TC-IC
SamplesSamplesBFU-E CFU4M CFU-GEMM BFU-E CFU-GM CFU-GEMM
CMLPB 17 65+3 3u~+3 1.3+0.215+3 B3~3 1.2+0.4
Normal PEla 23 74 ~ 3 24 + 2 2.2 1 0.3 11 + 2 89 + 2 0.5 + 0.2
NormalBMb 20 36+3 62~4 1.2+0.29+2 91 +2 0.8+0.3
a From 23
b From l~i
.
.
- ~ - ;
.
2080255
- 74 -
~able 6
Growth F~ctor Product~on by Retrovirally Infected M2-lOB4
Cell~
Bioactivity (ng/mL) in Medium
~.
Retroviral Ve~tor GN-CSF G-CS~ 3 ~-
.
~ninfectsd or N2 <O.Ol ~O.l <O.1
10 N2-tkG~-CSF 1.0 ~O.l <O.1
N2-tkG-CSF ~0.03 20 ~O.l
N2-tkIL-3 <O.Ol <O.1 6
:: .
.
'
2080255
Table 7
Comp~ri30n of the Content of Five-Week-Old Cocultures
Containing Differ0nt Type3 of or No Feeder (plastic)
~2-1084 ~uman MF Plastic
Nonndherent cells per 13 + 6 28 + 6* 1.2 + O.4*
cell pl~ted (no. (15) (13) (12)
experiments)
10 Clonogsnic cells per 4.8 + 1.0 6.2 ~ 1.1 0.10 + 0.03
100 cells plated
(no. sxperi~ent ) (15) (14) (15)
LTC-IC per 1,000 c~ll~ 2.3 ~ 0.4 3.4 + 0.8 0
plated (no. (12) (10) (4)
experim~nt~)
* Using a paired t-test a significant difference from ~2-
lOB4 was observed.
r~ :
'' ' ' : ~
.
.- . ' `. : ' ~ : '
.' ' ' "' ~'
', . '~ . ' ''' ''' ' ' ' ~ ~ ; .
~ ' ' ' .
2~0~55
_ 76 -
Table 8
Number of Clonogenic Progenitor~ per L~C-IC Harvested From
Five-Week-Old Cocultur~ Containing Different Types of
Feeder
~
No. of Clonogenic
Feeder Progenitor~ Per LTC-IC
Human MF 4.0 + 0.7
N2-lOB4 3.3 + 0.7
M2-lOR4 + G 5.5 ~ 1.0
N2-lOB4 ~ GM 6.4 + 2.1
M2-lOB4 + I~-3 2.8 + 0.8
M2-lOB4 + G + GN 3.7 ~ 1.1
N2-lOB4 + I~-3 + G 4.7 + 1.1
M2-lOB4 + Ih-3 + GM 4.3 + 1.0
M2-lOB4 ~ IS-3 + G + GM 6.0 + 2.4
_
Abbrevi~tions: G,G-CSF; GN,G~-CSF.
~' '': - ' : : -
2~025~
TABLE 9
Tablc 1. Quantl~tlon of LTC-IC and Clono~ lc Cells
In Nonnal PB
Cell l~pe Concentrat~on
IPer ml)
BFU-E 170 ~ 20
CFU-GM 51 ~ 5
CFU-GEMM 4.6 + 0.6
LTC-IC 2.9 + 0.5
.
Values for Indhrldual patlents were calculated by multlplying the progenltor frequency per
105 cells by the total nucleatcd cell recove~y aft both the T cell-depletlon and FH denslt~r
cent~ugatlon steps and then agaln ~y the WBC pcr ml. Values shown are the mean
SEM of data obtatned from 23 dlaerent normal Indhrlduals.
-: ~
.
- 78 - 2~8025~
TABLE 1 0
Table 2. Frequency. Enrlc~cnt and Recovy of LTC-IC and ClonogeDlc Cells In Va~ous
SubpopulaUon~ of the CD34+. T Cen-depleted Llght D~ty ~ractlon of Nomlal
Perlpheral 13boli Cells De~lned Acc~dlng to Uleir Exp~lon d HLA-DR
.
Cell ~pe Souroe F requencyb Er~mentC % Recove~d No. of
Evaluated ~ Exp
.
LTC-lCa ur~orted~ 0.0022 + 0.0004 - - 5
cells
DRhlgh f Og 5
DRlowf 3.7 ~ 1.11930 + 470 300 + 40 2
DR~h 1.0 _ 0.554~ ~ 270 48 + 36 3
DR I 2.8 + 0.51470 + 340 280 + 17 3
Clonogenlc unsorted~0. 1 1+ 0.02 - . 5
cells celh
DRhlgh t 7.6 + 0.4 65 + 22 2.7 + 1.2 4
DRIwf N.D,~
DR+h 21 ~ 2 240 ~ 40 21 + 6 4
DR I 15 + 4 160 + 40 38 + 5 4
,
a Measured as the total number of clonogenlc cells present after 5 weeks (I.e.. -~dC the absolute
LTC-IC number)
b Frequency of the cell typc evalualed ILTC-IC or total clonogenic ceDs) rclatlve to all nucleated
cells In the populatlon analyzed. fTo co~vert the LTC-IC frequencies shown to absolute
frequenclcs. dlvide by 4.1
c Calcula5ted by dhrldlng the progenltor frequency pcr 105 sorted ceL~ls by the progcnitor frcquency
pcr 10 unsorted, T cell-depleted. IUht-den~ty ceL~ In each lndhrldual acper~ment. and then
derivlng thc mcan _ SEM of these values for the number of c~cper~nent~ performed.
d Calcubted by mulUpb1ng thc percentage of cell~ rctrlcvcd In the fractlon Indlcated by the
correspondhg cakulated progenitor er~chmcnt for cach Indlvidual exper~nent ~dellned in
~ootnote c). and then derl~rtng the mcsn t SEM of thesc values for the number of e~cperlments
perfonned.
e Llght denslty. T cell-depleted cell~.
- 79 - 2080255
TABLE 10 cont ' d
f AS deflned In F'~gure 15C .
g Nonc dctccted: Le., d~.l5. Enrlcbment and mt~v~value~ therefore t cakulatçd.
h Refer~ to a ~ubpopulatlon o~ DR~ ceDs dcDned z~ fractlon 2 In F~gurc 17A.
Rcfers to a ~ubpopulauon of CD34+HI~-DE~1W cdb deflned as ~actlon 1 In ~gure 17A.
Not donc as a ~eparate measu~ment. DRlW ~ecovy ~ralues can bc ~nfased b~r addlng togethcr
values for DE~ and DP:.
.. , ?
,
, ~ .: - . ~ , : ... . . .
- 80- 208025~
TABLE 1 1
Relative Proportion~ of Dif ferent Type~ of Clonogenic Cell~
Detected Before and After S weeks in LTC ( 9~ of total ) .
Orlglnal No. of Clonogenlccellsa LTC-lCb
Progenltor Sampl~
Source
BFU-E C~V-Gbl CFU-CEMM BF'U-E CF~-GM CFI~-GEMM
Llght dcnslty
fractlon of
nonnal
bloodC 23 74 ~ 3 24 2 2.2 t 0.3 11 ~ 2 89 1 2 0.5 ~ 0.2
LTC-IC
enrlchcd
fractlon of
norrnal
bloodd 6 72 + 5 28~ 5 0.8*0.3 11~ 1 89~ 2 1.0 1 0.6
Nonnal
BMe 10 36~3 62~4 1.2 l 0.2 9~2 91 1 2 0.8~0.3
L rC -IC
er~ched
fractlon of
normal E3Me 10 24 ~ 5 75 ~ S 0.4 ~ 0.3 9 + 3 90 + 4 1.4 + 0.8
a Data shovm are the mcan ~ SEM of proportlonal values for spec1flc clonogenlc ccll types
expressed as a percent of aU clonogenlc cclls ILe.. E~FU-E plu~ CFU-GM plus CFU-GEMM~
measured ~n standard short-term mcthylce~ulosc assays.
b Data shown arc the mean * 5EM of proportlonal valucs for spcc~c clonogenlc ccD typcs
exprcsscd as a pc~lt of all clonogen~c ccDs ILe.~ ElFU E plus CFU-GM plus CFU-GEMM~
measurcd h me~hylcdlulose a~ay~ Or ccDs harvested rrom 5 weck-old LTC.
c Sarne samplcs as In Table 9 .
d Data rrom LTC IC In rractlon I ICD34~. DR ) ~n F4~urc l7 Ind~ a~d fractlon 1 ICD34~. Rh-
123dUII~ In F~gurt 18 (n=3) .
C Data ror nonnal BM rr~TI prr~ously publlsbed studlcs.20
