Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Hematopoietic Stem Cells
Field of the Invention
The present invention relates to human hematopoietic stem cells and to
methods of isolating and using such cells in compositions and in methods for
the reconstitution of a deficient or missing cell population. The present
invention also provides a population of human hematopoietic stem cells that
can be isolated and genetically altered for introduction in a human patient to
correct various genetic disorders.
Background of the Invention
The mammalian hematopoietic system consists of a heterogeneous array
of cells ranging from large numbers of differentiated cells with defined
function
to rare pluripotent stem cells with extensive developmental and proliferative
potential (1, 2, 3). The defining feature of a stem cell is its ability to
repopulate
the hematopoietic system of a recipient after transplantation. Stem cells are
playing an increasingly important role in clinical and commercial
applications,
as the role of stem cells in transplantation widens. Identification and
purification of stem cells is essential both to determine the cellular and
molecular factors that govern stem cell development and for the application of
clinical procedures including stem cell transplantation and gene therapy.
Cell surface expression of the CD34 antigen was thought to be the
distinguishing feature of stem cells because CD34 is downregulated as stem
cells differentiate into more abundant mature cells (4), and CD34 has been
used
as.a basis for isolation of stem cells. CD34, however, does not mark stem
cells
exclusively, since 1 % of bone marrow cells are CD34+ and include clonogenic
progenitors that are not able to repopulate the hematopoietic system after
transplantation. Other markers such as Thy-1 can be combined with CD34 to
positively select for a cell fraction more enriched in stem cells (S, 6, 7).
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Conversely, the CD34+ cell fraction can be enriched by eliminating cells that
express markers that are expressed on non-repopulating cells (e.g.lineage
antigens). Nevertheless, all current clinical and experimental protocols
utilizing
human stem cells, including ex vivo culture, gene therapy and bone marrow
transplantation, focus on CD34+ cells.
There have been reports (8, 9, 10) of murine CD34- hematopoietic stem
cells which are capable of long term repopulation. For human hematopoietic
stem cells, however, the CD34+ antigen has been regarded as a stem cell
marker without exception.
Summary of the Invention
The present inventors have identified and isolated a population of human
hematopoietic stem cells which do not express CD34 (CD34-), CD38 (CD38-)
or lineage specific markers (Lin-) and which are able to generate, by
I S proliferation and differentiation, multiple lineages of the human
hematopoietic
system, as evidenced by their ability to produce multilineage human
hematopoietic engraftment of immune-deficient NOD/SCID mice after
transplantation. Moreover, the repopulative capacity and the differentiative
capacity of the CD34-CD38-Lin- cells can be stimulated by in vitro culture of
these cells.
According to an object of the present invention there is provided a
substantially homogeneous population of human hematopoietic stem cells
which are CD34-Lin-.
According to another object of the present invention there is provided a
substantially homogeneous population of human hematopoietic stem cells
which are CD34-CD38-Lin-.
According to another object of the present invention there is provided a
therapeutic composition comprising an effective amount of CD34- Lin- human
hematopoietic stem cells and a pharmaceutically acceptable carrier.
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According to another object of the present invention there is provided a
therapeutic composition comprising an effective amount of CD34-CD38-Lin-
human hematopoietic stem cells and a pharmaceutically acceptable carrier.
According to yet a further object of the present invention there is
provided a method for obtaining a substantially enriched population of CD34-
Lin- human hematopoietic cells comprising the steps of:
- removing mononuclear cells expressing some lineage-specific antigens
from a sample of hematopoietic cells;
- combining the resultant hematopoietic cells with labeled antibodies to
which bind specifically to CD34+; and
- isolating the unbound CD34-Lin- cells.
According to still another object of the present invention there is
provided a method for reconstituting hematopoiesis in an immunocompromised
subject, the method comprising administering to a subject a composition
comprising an enriched population of CD34- Lin- stem cells.
According to a further object of the present invention there is provided a
method for introducing CD34-Lin- stem cells in a mammal, said method
comprising the steps of:
- providing an enriched population of CD34-Lin- stem cells; and
- introducing said stem cells into said mammal.
According to another object of the present invention there is provided a
method of treating a hematopoietic disorder in a subject, comprising:
- providing an enriched population of human CD34-CD38-Lin- stem
cells;
administering said stem cells to the subject in need of treatment.
According to another object of the present invention there is provided a
method for the production of CD34+ stem cells, said method comprising:
- providing an enriched population of human CD34-CD38-Lin- stem
cells;
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- culturing said stem cells in vitro under suitable conditions for a time
sufficient to allow said cells to differentiate into CD34+ cells; and
- isolating CD34+ stem cells.
According to still a further object of the present invention there is
provided a method for expanding a population of CD34-Lin- stem cells. said
method comprising the steps of:
- isolating CD34-Lin- stem cells from suitable hematopoietic source;
- culturing said isolated cells in vitro for a time sufficient and under
culture conditions to result in the expansion of said cells.
Brief Description of the Drawings
Certain embodiments of the invention are described, reference being
made to the accompanying drawings, wherein:
Figure lA shows cell surface expression of CD34 on cord blood cells
depleted for lineage markers (Lin-). (Panel I) Lin- cells were stained with a
class III monoclonal antibody for CD34 (581 ) conjugated to FITC (Becton
Dickinson, BD). Cells residing in R1 were considered CD34 negative (CD34-).
(Panel II) CD34- cells were purified using standard cell sorting techniques
and
re-analyzed using the same CD34-581 antibody. (Panel III and Panel IV)
Purified R1 cells were stained and re-analyzed using a class I monoclonal
antibody for CD34 (Immun-133) (Coulter) and a class II monoclonal antibody
for CD34 (Q-Bend-10) (Becton Dickinson. BD).
Figure 1B shows cell populations immunostained for CD34 expression.
Representative cells are shown from a total of 25-75 cells examined for each
treatment (n=2).
Figure IC shows a comparison of cell surface markers between CD34-
Lin- versus CD34+Lin- cord blood cells.
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Figure 2 shows level of human cell engraftment in NOD/SCID mice
transplanted with highly purified CD34-Lin- cells at various indicated doses.
Figure 3 shows the multiiineage differentiation of human CD34-Lin-
cells in NOD/SCID mice. Bone marrow from a highly engrafted mouse
transplanted with 120,000 CD34-Lin- cord blood cells was stained with various
human-specific monoclonal antibodies and analyzed by flow cytometry.
Approximately 106 mononuclear cells collected from mouse bone marrow
were prepared for rnultilineage analysis ( 12). (A) Cells with medium to high
forward scatter (region R1) were gated and further analyzed. (B) Histogram of
CD45 (pan-leukocyte marker) expression indicating that 2.5% of the cells
present in the murine bone marrow are human, gated R2. All further lineage
markers were examined on cells within gate R2 (CD45+). (C) Isotype control
for non-specific IgG staining of PE and FITC fluorescence. (D) Expression of
myeloid marker CD33 and granulocyte marker CD15; (E) pan-B cell markers
1 S CD 19 and CD20; (F) CD38 and the immature hematopoietic marker CD34; (G
and H) T-cell markers CD2, CD3, CD4 and CDB.
Figures 4A and 4B show frequency analyses of CD34-Lin- cells and
CD34-CD38-Lin- cells found in human hematopoietic tissue. Figure 4A:
Column I: Fetal liver collected from 8 week old human fetus, n=3; Column II:
Fetal blood aspirated from 19 week old fetus, n=1; Column III: Cord blood
collected from placenta at time of birth, n=3. Figure 4B: Column I: Normal
Adult Bone marrow, n=2; Column II: Bone marrow from a normal adult donor
after 5 days of G-CSF administration, n=2; Column III: Peripheral blood
collected from a normal adult donor after ~ days of G-CSF administration, n=2.
Figure 5 shows the percentage of input cells after in vitro culture of
CD34-Lin- cells. Purified cells were counted and seeded (250-2000) in wells
containing serum free media (SF) (solid bars) or SF supplemented with 25%
HUVEC- conditioned media ( shaded bars). Cells were counted each day and
the percentage of input cells was calculated ( n=3).
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Figure 6 shows an analysis of CD34 and CD38 expression of highly
purified cell populations after in vitro culture. A representative experiment
(n=4) of CD34 and CD38 cell surface expression performed on initially
purified CD34-CD38-Lin- cells, and purified cells after 2 and 4 days of
culture
in SF, SF supplemented with 5% FCS or 25% HLTVEC-CM. The entire
contents of individual wells was collected at 2 and 4 days (5000-10, 000
cells),
stained with monoclonal antibodies CD34 and CD38 directly conjugated to
FITC and PE respectively (Becton Dickinson, BD). Stained populations were
then washed and analyzed using standard flow cytometric techniques, followed
by the display of histograms using the Cell Quest software program (BD).
Figure 7A shows levels of engraftment of non expanded CD34-CD38-
Lin- or CD34-CD38+Lin- cell fractions in NOD/SCID mice.
Figure 7B shows the capacity of CD34-Lin-, CD34-CD38+Lin- and
CD34-CD38-Lin- cell fractions to engraft NOD/SCID after ex vivo culture. (A)
The level of human cell engraftment is the BM of 136 mice transplanted with
either CD34-Lin-, CD34-CD38-Lin- or CD34-CD38+Lin- cell fractions seeded
at the indicated cell doses after day 2 or 4 of ex vivo culture from 43 CB and
3BM samples.
Figures 8A to 8H show the multilineage differentiation of human CD34-
Lin- cells in NOD/SCID mice after ex vivo culture represented in histograms.
Figure 8A: Histogram of CD45 (human-specific pan-leukocyte marker)
expression indicating that 7% of the cells present in the murine bone marrow
are human. Figure 8B: Forward and Side scatter of the CD45 human cells.
Subsequent analysis of lineage markers was done on CD4~+ cells within gate
R1 (lymphoid and blast cells) or R2 (myeloid cells) gates. Figure 8C: Analysis
for the presence of immature cells using the CD34 and CD38 markers. Figure
8D: Analysis for the presence of human B cell lineage cells using CD 19 and
CD20 markers. Figures 8E to 8F: Analysis for the presence of human T
lymphocytes using the panel of T cell markers: CD2, CD3, CD4 and CDB.
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Figures 8G to 8H: Analysis for the presence of myeloid cells using CD33,
CD 14, CD 15 and CD 13 markers.
Figure 9 shows DNA analysis of NOD/SCID mice transplanted with
CD34-Lin- or CD34+CD38-Lin- cells purified from the same cord blood
sample and cultured in the presence of 5% FCS. The southern blot was
hybridized with a human chromosome 17-specific a-satellite probe.
Detailed Description of the Invention
The identification of the CD34-Lin- and CD34-CD38-Lin- human
hematopoietic stem cell has important implications for understanding the
origin
of hematopoietic diseases such as leukemia and for clinical procedures such as
stem cell transplantation and gene therapy for the treatment of various
diseases.
The stem cell of the present invention is also important for the treatment or
prophylaxis against disease or infection, for the reconstitution of deficient
or
i 5 missing cell populations, as for example in cancer patients after
myeloablative
therapy, and for the treatment of congenital or acquired genetic abnormalities
and defects by the introduction of desired genetic information into the
patient.
The CD34-Lin- and CD34-CD38-Lin- human hematopoietic stem cells
of the present invention can be isolated using standard techniques of cell
sorting
in order to rapidly identify and isolate the cells so that they may be
isolated ex
vivo and cultured in vitro to provide an expanded population of the cells for
use
as a therapeutic composition for humans requiring such. Such ex vivo cultured
cells may be genetically manipulated as required prior to being reintroduced
back into a patient.
In accordance with one embodiment, the invention provides a
substantially homogeneous population of human hematopoietic stem cells
which are characterized as CD34-Lin- and CD34-CD38-Lin-.
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In accordance with a further embodiment. the invention provides a
method for preparing a substantially homogeneous population of human
hematopoietic stem cells which are CD34-Lin- and CD34-CD38-Lin-.
In accordance with a further embodiment, the invention provides a
S therapeutic composition comprising CD34-CD38-Lin- human hematopoietic
stem cells. The invention also provides a therapeutic composition comprising
CD34-CD38-Lin- human hematopoietic stem cells. Such compositions can be
used to treat hematopoietic disorders such as malignancies (i.e. leukemia),
immune disorders and diseases resulting from a failure or a dysfunction of
normal blood cell production or maturation. Diseases may include but are not
restricted to congenital disorders, severe combined immunodeficiency,
Wiskott-Aldrich syndrome, Fanconi's anemia, congenital red cell aplasia,
lysosomal storage disease, thalassemia major, sickle cell anemia, aplastic
anemia, acute lymphoblastic leukemia, acute myelogenous leukemia,
1 S megakaryoblastic leukemia, hematologic melanomas, lymphoma, multiple
myeloma, myelodysplastic syndromes, carcinomas, neuroblastomas, arthritis
and neurological genetic diseases (e.g. Gaucher Disease).
The novel stem cells of the present invention may be used in various
clinical procedures such as stem cell transplantation for the reconstitution
of a
deficient or a missing cell population, therapy, gene therapy and for
combating
infection.
In accordance with another aspect of the invention there is provided a
method for the treatment of hematopoietic disorders such as leukemia
comprising the use of human hematopoietic cells characterized as CD34-Lin-
and CD34-CD38-Lin-.
In accordance with another aspect of the present invention is a method
for the ex vivo generation of human hematopoietic cells using CD34-Lin- cells
and/or CD34-CD38-Lin- cells.
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In accordance with another aspect of the present invention is a gene
therapy method for providing genetically altered human hematopoietic cells
characterized as CD34-Lin- or CD34-CD38-Lin- to a patient.
In accordance with another aspect of the present invention is a method
for reconstituting deficient or missing human hematopoietic cell populations
comprising the use of CD34-Lin- cells or CD34-CD38-Lin- cells.
In accordance with another aspect of the present invention is a method
for transplanting human hematopoietic cell populations to a patient comprising
the use of CD34-Lin- and CD34-CD38-Lin- cells.
In accordance with another aspect of the present invention is a method
for combatting infection in a patient comprising administering an effective
amount of human hematopoietic cells characterized as CD34-Lin- or CD34-
CD3 8-Lin-.
In accordance with yet a further aspect of the present invention there is
provided a method utilizing human hematopoietic cells characterized as CD34-
Lin- or CD34-CD38-Lin- for the production of CD34+ human hematopoietic
cells.
In accordance with a further aspect of the present invention is a method
for increasing the repopulating capacity of human hematopoietic cells
characterized as CD34-Lin- or CD34-CD38-Lin- by culturing such cells irZ
vitro for several days.
In accordance with a further aspect of the present invention is a method
for screening candidate compounds affecting proliferation or differentiation
of
stem cells characterized as CD34-Lin- and CD34-CD38-Lin-.
Characterization of Hematopoietic Stem Cells
It was determined, using CD34 class III fluorescent monoclonal
antibodies, that CD34- cells that do not express lineage markers exist in
human
hematopoietic tissues (Fig. 1 A). The possibility that CD34 protein was
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produced in the CD34-Lin- cells but not transported to the cell surface was
excluded using permeabilized, stained cytospins of purified CD34-Lin- that
were further conjugated with fluorescent monoclonal antibodies. This
confirmed that no CD34-FITC signal was detected in the CD34-Lin- cells (Fig.
1 B).
Heterogeneity within human CD34+Lin- cells is well documented and
further subdivision for the most primitive cells is typically based on the
cell
surface markers CD38, c-kit, Thy-1 and HLA-DR. The expression of these
markers on both the CD34+Lin- and CD34-Lin- cells was compared (Fig. 1 C).
CD34-Lin- cells displayed a bi-modal distribution of CD38, clearly dividing
the
population into two fractions, in contrast to the high proportion of CD34+Lin-
cells that express CD38 (Fig. 1 C). Cell surface expression of c-kit was
similar
between the two populations, while CD34-Lin- cells were almost exclusively
Thy-1- and HLA-DR- (Fig. 1C). Both the absence ofHLA-DR expression and
the presence of Thy-1 have been proposed as defining more primitive
subfractions within the CD34+Lin- population. Therefore, the CD34-Lin-
population derived from cord blood is a distinct population which differs from
primitive CD34+Lin- cells not only in CD34 expression but also in phenotypic
heterogeneity based on additional stem-cell associated markers.
Using clonogenic methylcellulose assays, the hematopoietic progenitor
activity of CD34-Lin-, CD34-CD38-Lin- and CD34-CD38+Lin- cells was
determined by comparing their CFC and LTC-IC content. The clonogenic
capacity of CD34-Lin- cells was extremely low in comparison to that of
CD34+CD38-Lin- cells (Table I). As many as 10,000 cells needed to be seeded
on MS-5 stroma to detect a single LTC-IC within the CD34-Lin- cell fraction,
while further purification demonstrated that detection of LTC-IC in the CD34-
CD38-Lin- fraction required seeding of at least 2000 cells. By contrast, as
few
as 10 CD34+CD38-Lin- cells contain an LTC-IC. The CD34-CD38+Lin- cells
were devoid of LTC-IC activity (limit of detection at 10,000 cells) but
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contained a much higher capacity to form CFC (colony-forming cells)
specifically committed to the erythroid lineage (Table I). The low efficiency
of
production of myeloid and erythroid committed progenitors, as well as the more
primitive LTC-IC, were similar to observations made with murine CD34-Lin-
cells in the same assay systems and suggested that functional similarities may
exist between the CD34-Lin- cells from these two species.
The only conclusive method for detecting stem cells is to determine their
ability to repopulate recipient hosts. The repopulation capacity of primitive
human cells can be assayed by their ability to initiate multilineage human
engraftment in immune-deficient NOD/SCID mice. Based on cell purification
and gene marking, cells capable of repopulating NOD/SCID mice (termed the
SCID-Repopulating Cell, SRC) were established as distinct from, and more
primitive than, the majority of progenitors detected in in vitro assays.
CD34+CD38-Lin- cells, and not CD34+CD38+Lin- cells, gave rise to
multilineage engraftment. Transplantation of as many as 106 CD34-Lin+ cells
did not give engraftment. To determine whether highly purified CD34-Lin-
cells had SRC activity, and to determine the frequency of any repopulating
cells, CD34-Lin- cells were transplanted at varying cell doses into NOD/SCID
mice using standard protocols, and bone marrow was analyzed for the presence
of human cells after 8-12 weeks. The level of human cell engraftment in 23
NOD/SCID mice was quantitated by FACS and DNA analysis for the presence
of human cells and results are summarized in Fig. 2. A large proportion of
transplanted mice were engrafted with human cells, indicating that CD34-Lin-
cells were able to repopulate NOD/SCID mice. This cell was designated the
.CD34NEG-SCID Repopulating Cell (CD34NEG-SRC). The frequency was 1
CD34NEG-SRC in 12,000 CD34-Lin- cells. The differentiative and
proliferative capacity of the CD34NEG-SRC cell was assessed by flow
cytometric analysis. A representative engrafted NOD/SCID mouse 10 weeks
after the transplant of 120,000 CD34-Lin- cells is shown in Fig. 3. Cells with
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medium to high forward scatter (region Rl, Fig. 3A) were gated and further
analyzed, based on CD45 expression, a human specific pan-leukocyte marker
(Fig. 3B). The isotype control is shown in Fig. 3C. The bone marrow of this
mouse contained 2.5% CD45+ human cells (Fig. 3B), or at least 106 total
S human cells indicating that the CD34-Lin- cells have extensive proliferative
capacity. Human granulocytes (CD 1 S+) were present among myeloid cells
(CD33+) (Fig. 3D). Human B-lymphoid cells were also present in the murine
bone marrow as shown by staining for CD19 and CD20 (Fig. 3E). Interestingly,
human T-cells expressing both CD2 and CD3 (Fig. 3G), along with CD4 and
CD8 positive cells (Fig. 3H), were also identified. NOD/SCID mice
transplanted with highly purified primitive CD34+CD38-Lin- cells never gave
rise to engraftment containing T-cells demonstrating the unique in vivo
repopulation behavior of the CD34-Lin- cells. In addition to multilineage
engraftment, immature CD34+ and CD34+CD38- cells were detected (Fig. 3F).
It was concluded that human CD34-Lin- cells have the ability to repopulate
NOD/SCID mice and differentiate in vivo into multiple lineages of myeloid and
lymphoid cells. The production of CD34+CD38- and CD34+CD38+ cells in
vivo suggests that CD34-Lin- cells are developmentally earlier than CD34+
cells in the hierarchy of human hematopoiesis.
There is evidence that the frequency of primitive cells changes during
ontogeny with the highest proportions seen in the fetus. A variety of fetal,
neonatal, and adult sources of human hematopoietic tissue were analyzed in an
attempt to identify and quantify the CD34-Lin- population. The results
indicate
(Fig. 4A,4B) that CD34-Lin- cells are produced early in human ontogeny and
can persist throughout adult life and that the mechanisms that operate during
the mobilization of CD34+ cells by G-CSF also affect CD34-Lin- cells.
This data provides the first identification of a novel human
hematopoietic stem cell that does not express CD34 or lineage-specific
markers. As determined by all available monoclonal antibodies, this population
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is not only distinct by the absence of CD34, but also by the lack of HLA-DR
and Thy-1 markers. In addition to these phenotypic differences, several lines
of
evidence functionally distinguish these two stem cell populations. While the
CD34-Lin- cells have limited hematopoietic activity in vitro, CD34+CD38-Lin-
cells are highly clonogenic based on their ability to produce CFC and LTC-IC.
Although both stem cell fractions are capable of repopulation, the presence of
T-cells within the multilineage engraftment is a unique characteristic of CD34-
Lin- transplantation; human T cells have not been detected in mice
transplanted
with CD34+CD38-Lin- cells (n=25). It is unlikely, therefore, that NOD/SCID
repopulation was derived from contamination of CD34-Lin- cells by
CD34+CD38-Lin- cells. Furthermore, based on LTC-1C frequency, a minimum
of 1 LTC-IC resides within 10 highly purified CD34+CD38-Lin- cells. In
contrast to flow cytometry, by which it has been determined that the CD34-Lin-
population is 99% (or in some cases 100%) pure, the LTC-IC assay allows
detection of a smaller number of contaminating CD34+CD38-Lin- cells. Using
this assay, only a single LTC-IC could be detected in as many as 10 000 CD34-
Lin- cells. If this LTC-IC activity came from a CD34+CD38-Lin- cell, a
maximum of 10 CD34+CD38-Lin- cells could be contained in the CD34-Lin-
purified fraction (0.1 % contamination). In addition, repopulated mice have
not
been observed when only 10 CD34+CD38-Lin- cells were transplanted (12).
Since the frequency of SRC derived from CD34-Lin- cells is 1 in 125 000, a
maximum of 125 CD34+CD38-Lin- cells could have been transplanted, again
less than the number needed to repopulate CD34+CD38- cells.
The in vivo differentiation of human CD34-Lin- cells into CD34+ and
lineage positive cells after murine engraftment suggests that CD34-Lin- cells
preceed CD34+ cells in the hierarchy of human hematopoiesis.
It is also possible that the CD34-Lin- cells upon further phenotypic
evaluation may contain certain subpopulations of cells with have further
different phenotypic antigenic expression. It is also possible that CD34-Lin-
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cells have further phenotypic antigenic expression themselves that will
further
help to isolate these new class of stem cells in a more efficient and rapid
manner.
The identification of these novel repopulating cells, termed
CD34"e°°-
SCID repopulating cells (CD34"eg-SRC), provides an opportunity to examine
the differentiation and proliferation potential of these cells and to
establish their
relationship to other cells within the human stem cell hierarchy. These
CD34"e~-
SRC are found within both the CD34-Lin- cell fraction and the CD34-CD38-
Lin- fraction. CD38+ cells identified within the CD34-Lin- fraction do not
have repopulation potential. .
Many studies based on ex vivo culture have demonstrated that there is
heterogeneity within the CD34+ cell fraction. Subfractionation of the CD34+
cells on the basis of Thy I , CD38, and HLA-DR expression together with in
vitro clonogenic and LTC-IC assays have demonstrated the progenitor-progeny
relationship of the various cell types that make up the stem cell hierarchy
(4,
14). The availability of the SRC assay to detect even earlier cell types has
added more information about the organization of cells within this hierarchy
(14). Moreover, it was demonstrated that the SRC (derived from CD34+CD38-
cells) can be expanded for 4 days in serum-free cultures without inducing
their
differentiation. However, all SRC are lost within an additional 4 days of
culture concommitant with the appearance of more differentiated CD38+ cells
( 11 ). At the same time, both colony-forming cells (CFC) and long-term
culture
initiating cells (LTC-IC) could be greatly expanded during 8 days of culture,
demonstrating that the majority of the SRC are a distinct population ( 11 ),
but
may be closely related to ELTC-IC ( 18). Thus, in vitro culture systems can be
used to identify very fine transitions in the developmental program by
combining both flow cytometry and functional CFC, LTC-IC and SRC assays.
It is now demonstrated that ex vivo culture of CD34-Lin- cells can induce the
appearance of CD34+ cells and can increase the proportion of CD34-Lin- cells
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that have SRC activity. These studies provide new insight into the
developmental program of human hematopoietic stem cells.
Earlier studies demonstrated that the CD34-Lin- cell fraction expressed a
bimodal distribution of CD38, allowing for further purification into CD38- and
CD38+ subpopulations (12). To determine whether the CD34-Lin-, CD34-
CD38-Lin-, and CD34-CD38+Lin- cells could be induced to proliferate and/or
differentiate, these cells were cultured in defined serum-free (SF) conditions
that have been previously shown to support expansion of Lin34+38- cells and
maintenance and modest increase of POS CD34 POS-SRC (11, 17). Cells were
plated in methlycellulose assays at day 0, and after day 4 of liquid culture,
(Table II) to determine the effect of cytokine stimulation on the clonogenic
progenitors present in CD34-Lin-, CD34-CD38-Lin- and CD34-CD38+Lin-
cell fractions. Both the CD34-Lin- and more purified CD34-CD38-Lin- cells
have a low plating efficiency (PE), 1 in 89 and I in 297 CFC respectively,
whereas a higher PE of CD34-CD38+Lin- cells (1 in 10.4 cells) was seen.
Interestingly, the clonogenic capacity of the CD34-CD38+Lin- cells was
restricted to the erythroid lineage. After 4 days of culture in SF media, or
SF
media supplemented with the addition of 25% conditioned medium obtained
from primary human umbilical vein endothelial cells (HUVEC-CM), the PE of
all the sub-populations examined had decreased, whereas the addition of 5% of
FCS .increased the clonogenicity of CD34-Lin- cells and, to a greater extent,
CD34-CD38+Lin- cells {Table II). This difference in clonogenicity may reflect
heterogenity within CD34-Lin- cells and demonstrates that the CD38+
subfraction is already committed to the erythroid lineage, suggesting that the
CD34neg-SRC resides in the CD34-CD38-Lin- subfraction.
To determine whether the CD34-CD38-Lin- cells could be stimulated to
proliferate, changes in cell number were recorded between day 0 and 4 of
culture with SF or 25% HUVEC-CM (Fig. 5). The total number of cells
decreased by 2 fold at day 4 in SF media. Supplementation of 5% fetal calf
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serum (FCS) showed no increase in the viability of these cells (data not
shown).
However, the addition of HUVEC-CM to SF media maintained or slightly
increased the total cellularity (Fig.S). These results demonstrate that
culture
conditions that are optimal for CD34+CD38-Lin- cells ( 11 ) are unable to
support CD34-CD38-Lin- cells, while soluble components present in primary
HUVEC-CM seem to permit their survival.
The effect of culture on the differentiatiation program of CD34-CD38-
Lin- cells, from individual wells was analyzed by flow cytometry after 2 and 4
days of culture in various conditions (Fig. 6). Surprisingly, CD34-CD38-Lin-
cells seeded in SF media began expression of CD34, which could be enhanced
with the addition of 5% serum (Fig. 6). In contrast, the majority of cells
obtained after 2 or 4 days of culture in the presence of 25% HUVEC
conditioned medium still maintained the original CD34-CD38-Lin- phenotype.
The stimulation of CD34-CD38-Lin- cells to differentiate and produce
CD34+CD38- cells suggests that CD34-CD38- cells precede the CD34+CD38-
population in the hierarchy of human hematopoiesis. Moreover, these results
indicate that the CD34-CD38-Lin- cells respond to signals present in SF or 5%
serum conditions and that HUVEC-CM can inhibit this stimulation.
To confirm which fraction contained CD34neg-SRC cells, both CD34-
CD38-Lin- and CD34-CD38+Lin- cells were transplanted into NOD/SCiD
mice. Transplantation with as few as 10,000 or 4,000 CD34-CD38-Lin- cells,
derived from cord blood or bone marrow respectively, resulted in engraftment
(Fig. 7A), whereas as many as 180,000 CD34-CD38+Lin- cells were incapable
of repopulation (Fig 7A). These data indicate that the CD34neg-SRC cells
present in the CD34-Lin- fraction are restricted to the CD38- subfraction.
However, since an entire cord blood sample contains only 1 or 2 CD34neg-
SRC {e.g. frequency is I CD34neg-SRC in 125.000 CD34-Lin- cells and one
cord blood sample contains up to 250,000 CD34-Lin- cells), the losses
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associated with subselecting based on CD38 expression resulted in only 9% of
samples which repopulated NOD/SCID mice.
To evaluate the repopulating activity of CD34-Lin- and more highly
purified CD34-CD38-Lin- cell fractions after ex vivo culture, cultured cells
were injected into NOD/SCID mice and the level of human engraftment
evaluated after 8 to 10 weeks. Purified cell fractions from 44 CB and 3 BM
samples cultured for 2 and 4 days in SF, SF supplemented with 5% FCS or 25%
HUVEC-CM were transplanted at various doses into 144 recipient NOD/SCID
mice and the level of human cell engraftment was determined (Figure 7B). A
total of IS of 35 mice were engrafted following transplantation with CD34-Lin-
cells that had been cultured for 4 days in SF or S% FCS at cell doses below
the
calculated frequency of CD34-SRC. For example, 13 of 29 mice were
engrafted following transplantation of 50,000 to as few as 4,000 cultured
CD34-Lin- cells. By contrast. only 1 of 7 mice were engrafted when 100,000
uncultured CD34-Lin- cells were transplanted (Figure 2). Similarly, a higher
proportion of mice (33%) transplanted with cultured CD34-CD38-Lin- cells
were engrafted (Figure 7B) compared to mice transplanted with similar doses
of uncultured CD34-CD38-Lin- (Figure 7B). Quantitative analysis using
Poisson statistics indicated a frequency of 1 CD-SRC in 38,000 cultured CD34-
CD38-Lin- cells (range 1 /22,000 to I /71,000). It was not possible to
calculate
the frequency of CD34-SRC in uncultured CD34-CD38-Lin- cells (Figure 7A)
because there was infrequent engraftment despite injection of as many as
50,000 cells. This result indicates that the actual stem cell frequency of
freshly
isolated cells must be much lower compared to cultured cells. The human
lineage distribution in mice transplanted with expanded CD34-Lin- and CD34-
CD38-Lin- cells is similar to that with unstimulated purified CD34-Lin- cells
(Figure 8). In addition, the inability of cultured CD34-CD38+Lin- cells to
engraft mice confirms the absense of repopulating cells within this fraction.
It
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was evident that culture conditions, especially HLIVEC-CM, induce an increase
in the number of CD34-SRC in the absence of cell proliferation.
The bone marrow of engrafted mice was analyzed by multiparameter
flow cytometry to determine whether cultured CD34-Lin- repopulating cells
possessed the same in vivo proliferative and differentiative capacity as
uncultured cells. A representative analysis of the bone marrow of a NOD/SCID
mouse transplanted with an initial population of 40,000 CD34-Lin- cells after
4
days of culture is shown in Figure 8. The bone marrow of this mouse contained
7% human cells as detected by expression of CD45, a human specific pan-
leukocyte marker (Fig. 8). Both B and T-lymphoid cells were present in the
murine bone marrow as shown by staining for CD19, CD20 and CD4, CD3
antigens (Fig. 8D-F). The presence of CD3 3+, CD 14+, CD 1 S+ and CD 13+
cells indicated the differentiation potential of CD38-CD38-Lin- cells to the
myeloid Iineages (Fig. 8G-H). The engraftment pattern of mice transplanted
with expanded CD34-CD38-Lin- cells is similar to that observed with
unstimulated purified CD34-Lin- cells. The presence of human T-cells is a
unique feature of CD34-Lin- engraftment, since T-cells have not been detected
in mice transplanted with purified CD34+CD38-Lin- cells either before or after
ex vivo culture.
We had previously found that the CD34+SRC are lost if CD34+CD38-
Lin- or CD34+Lin- cells are cultured in the presence of serum suggesting that
the CD34-SRC are biologically distinct from CD34+SRC. To test this directly,
CD34+CD38-Lin- and CD34-Lin- cells from the same human CB sample were
cultured for 4 days under the dame serum containing conditions. In a
representative experiment, 3 out of 6 mice were engrafted following
transplantation with cultured CD34-Lin- cells (Figure 9). In contrast, 5,000
and
10,000 CD34+CD38-Lin- cells, containing 10-20 CD34+SRC, cultured under
the same conditions were unable to engraft NOD/SCID mice. These results
provide independent confirmation that the repopulating cells derived from the
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CD34-Lin- subfraction are biologically distinct from those derived fTOm
CD34+CD38-Lin- cells.
It is believed that the repopulating cells of the human hematopoietic
system are the CD34-CD38-Lin- sub-population and short term ex vivo culture
of this fraction has been observed to increase the proportion of repopulating
cells. The ability of CD34-CD38-Lin- cells to produce CD34+CD38- cells in
vitro and in vivo demonstrates the developmental capacity of these cells and
further suggests that this population of cells is more primitive than the CD34
positive fraction. In addition, conditions evaluated here provide the
foundation
for future gene transfer and ex-vivo expansion of this novel population and
for
the identification of factors that stimulate their proliferation and
differentiation.
Furthermore, the knowledge that CD34-Lin- cells exist and can
repopulate the human hematopoietic system provides a novel therapeutic
composition and a method for the treatment of hematopoietic disorders. In
particular, the composition and method can be used to treat hematopoietic
disorders such as leukemia and for several clinical procedures such as stem
cell
transplantation, therapy, for combatting infection and for cell
reconstitution.
These cells can also be used to generate CD34+ cells and possibly other cell
types.
The new class of stem cells of the present invention provide a new
method of treatment for various disorders that is very advantageous. From a
small sample of bone marrow, peripheral blood or cord blood, the CD34-Lin-
cells can be isolated, maintained in culture, enriched and expanded and stored
for later use or further stimulated in culture to differentiate and provide
other
sources of cell types.
It is understood by those skilled in the art that the stem cells of the
present invention can be identified and isolated from bone marrow, peripheral
blood and cord blood. The most clinically advantageous source is peripheral
blood due to the fact that the procedure for obtaining such is easy and non-
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invasive. Collection of peripheral blood also has no health effect on the
donor:
While peripheral blood is the most convenient and least invasive source for
use
in isolating the stem cells of the present invention, it is understood by
those
skilled in the art that the bone marrow and cord blood are more ideal as a
starting point due to the larger percentage of stem cells present in such.
The stem cells of the present invention can be isolated using standard
techniques known in the art to identify classes of cells and to select subsets
of
cells of the hematopoietic systems. One such technique is flow cytometry. A
flow cytometer can identify different cells by measuring the light they
scatter or
the fluorescence they emit as they flow through a laser beam. This it can sort
out cells of a particular type from a mixture. A fluorescence activated cell
sorter or FACS, can select one cells from thousands of other cells. FACS
utilizes a multicolour flow cytometer to detect and separate cells bound with
fluorescent conjugated antibodies to the specific antigens that identify the
development stage or lineage stage of the cells of the hematopoietic system.
Detectable fluorescent signals are generating by hitting the cells with a
laser
beam as they pass through a flow sheath. A nonfluourescent forward scatter
signal is used to represent volume and a side scatter signal detects cellular
texture and granularity. The colour signals of the fluorochromes used to
conjugate with the antibodies detect the cell specific antigens. FACS analysis
can analyze two or more colours simultaneously and generate data which is
inputted into a computer program to generate colour plots, histograms and
perform statistical analysis.
The stem cells of the present invention can also be isolated using a
method of negative selection by depletion of lineage positive cells. In this
technique, the undesired cells are selected for and depleted leaving behind
the
desired cells.
Other techniques which may be used to isolate the stem cells of the
present invention include immunoseparation where antibodies against specific
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receptor molecules are used together with immunoaffinity columns to bind cells
having the specific target receptor. The targeted cells are then removed from
the antibody complex by the use of shear fluid force.
The cells of the present invention can be used to screen compounds
S which may affect their proliferation and/or differentiation into other cell
types.
For example, an isolated population of CD34-Lin- cells may be suitably
cultured in vitro to which selected growth factors, cytokines, chemicals,
peptides and other agents are added individually or in specific combination.
One skilled in the art would readily comprehend the conditions and procedures
for preparing such a cell culture and the amounts of agent to add for such
testing. After a period of time the CD34-Lin- cells may be phenotypically
characterized and counted in order to determine the effect of the added
agent(s).
In this manner, one may establish a simple method for producing a specific
cell
type for a clinical application.
The cells of the present invention can be used for understanding the
origin of hematopoietic diseases such as leukemia and for clinical procedures
such as stem cell transplantation and gene therapy for the treatment of
various
diseases. The stem cell of the present invention is also important for the
treatment or prophylaxis against disease or infection, for the reconstitution
of
deficient or missing cell populations, as for example in cancer patients after
myeloablative therapy, and for the treatment of congenital or acquired genetic
abnormalities and defects by the introduction of desired genetic information
into the patient.
In an embodiment of the present invention, the CD34-Lin- or CD34-
CD38-Lin- stem cells can be utilized for stem cell transplantation in order to
reconstitute missing or deficient cell populations. Bone marrow transplants
are
typically done in order to restore hematopoiesis in cancer patients receiving
high doses of chemotherapy and/or radiation therapy as well as in leukemia
patients and aplastic anemia patients. Cord blood has recently been used in
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order to reconstitute hematopoiesis as an alternative to bone marrow
transplants. However, there are several disadvantageous with bone marrow
transplants as they are highly invasive and require major surgery. One also
must find a suitable phenotypically matched donor. The patient's body must be
rid of all tumor cells which involves the use of cytotoxic chemicals leaving
the
patient hematopoietically deficient. A source of bone marrow sample is then
engrafted after which it takes 3 to 4 weeks for engraftment to occur.
In contrast to bone marrow transplants, the present invention allows one
to identify and purify CD34-Lin- or CD34-CD38-Lin- cells from a suitable
source such as bone marrow, cord blood or peripheral blood. These cells can
then be enriched ex vivo and transplanted or infused back into a patient
missing
or deficient in a cell population. The cells may also be treated genetically
or
chemically prior to transplantation back into the patient. The procedure for
reinfusion of the stem cells is less harmful to a patient than the
reintroduction of
1 S bone marrow and requires a substantially smaller volume of cells than bone
marrow transplants. It is understood by those skilled in the art that the
cells of
the present invention can be differentiated into various types of mature cells
which may be used for transplantation.
Peripheral blood transplantation can also be used to isolate and provide
back an enriched culture of CD34-Lin- or CD34-CD38-Lin- cells.
The invention provides a therapeutic composition comprising CD34-
Lin- human hematopoietic stem cells. The invention also provides a
therapeutic composition comprising CD34-CD38-Lin- human hematopoietic
stem cells. Such compositions can be used to treat hematopoietic disorders
such as malignancies (i.e. leukemia), immune disorders and diseases resulting
from a failure or a dysfunction of normal blood cell production or maturation.
Diseases may include but are not restricted to congenital disorders, severe
combined immunodeficiency, Wiskott-Aldrich syndrome, Fanconi's anemia,
congenital red cell aplasia, lysosomal storage disease, thalassemia major.
sickle
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cell anemia, aplastic anemia, acute lymphoblastic leukemia, acute myelogenous
leukemia, megakaryoblastic leukemia, hematologic melanomas, lymphoma,
multiple myeloma, myelodysplastic syndromes, carcinomas, neuroblastomas,
arthritis and neurological genetic diseases (e.g. Gaucher Disease).
It is understood by those skilled in the art that the compositions of the
present invention may comprises substantially pure populations of CD34- Lin-
human hematopoietic stem cells or CD34-CD38-Lin- human hematopoietic
stem cells. The compositions may also comprise enriched cultures of CD34-
Lin- or CD34-CD38-Lin- cells. Whether substantially pure or enriched, the
compositions of the present invention may additionally comprise cells selected
from the group consisting of CD34+ cells, Thy-1 cells, CD4+ cells, CD56+
cells, CD33+ cells, CD9+ cells, CD11+ cells, CD41+ cells, CD45 cells and
mixtures thereof. The type of composition made depends on the end use. For
example, for the treatment of leukemia it is desired to provide a composition
1 S comprising substantially homogenous populations of CD34-Lin- or CD34-
CD38-Lin- cells whereas for a standard stem cell transplant for other cancers
or
autoimmune diseases, the composition may comprise a mixture of CD34-Lin-
or CD34-CD38-Lin- cells together with CD34+ cells.
It is also possible to utilize as therapeutic compositions substantially
enriched populations of cells characterized phenotypically as CD34-Lin- or
CD34-CD38-Lin- but which also may be further phenotypically characterized
by the presence or absence of other antigenic markers.
It is also within the scope of the present invention to produce a
composition comprising CD34-Lin- or CD34-CD38-Lin- cells to which
infection fighting cells (B lymphocytes, T lymphocytes and their precursors)
blood clotting cells (platelets), or any of the myeloid lineage cells
(erythrocytes,
granulocytes, macrophages, monocytes, basophils, eosinophils and their
precursors) may be added to provide a "specialized" composition specific for a
particular disease. It is also understood by those skilled in the art that
various
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pharmaceutical agents may be added to the compositions of the invention in
order to treat specific disease states. For example, such pharmaceutical
agents
may include antibiotics, chemotherapeutic agents, cytokines, etc.
The stem cells of the present invention can be used to fight against both
disease and infection. Cell populations to be transplanted can be screened and
treated for infections such as viral (e.g. AIDS, herpes, hepatitis, etc.,),
bacterial
(e.g. staphylococcus, streptococcus, etc.,) and fungal in vitro. A sample of a
patient's blood or bone marrow containing the infected cells can be purged of
infection and the stem cells isolated and enriched ex vivo. In order to
destroy
the bacteria or virus which may affect such cells, the cultured cells may be
treated with a suitable antiviral agent or antibacterial agent. Alternatively,
the
isolated cells can be individually screened to select for uninfected cells and
the
resulting population enriched. Prior to the implantation of the treated or
untreated selected uninfected cells, the patient to receive such transplant is
treated with a suitable chemical agent, drug or radiation treatment to
eliminate
all infected cells. The enriched sample of stem cells can then be transplanted
into the patient. If desired, prior to transplantation into the patient, other
cell
types may be added to the culture of stem cell's depending on the disease
condition to be treated. For example, in the case of AIDS, a fresh supply of
uninfected T-cells may also be added to the enriched culture of CD34-Lin- or
CD34-CD38-Lin- cells.
In another embodiment, the CD34-Lin- or CD34-CD3 8-Lin- cells may
be used as a method of gene therapy. The CD34-Lin- or CD34-CD38-Lin-
cells may be isolated and enriched in in vitro culture where a desired genetic
2~ sequence can be inserted into the cells prior to their reintroduction into
a
patient. The genetic element introduced can simply be one to correct a defect
in the cells themselves or to target a specific recombinant gene sequence to a
specific area of the patient. Examples of diseases which may be treated with
genetically altered stem cells of the present invention include but are not
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restricted to hemophilia A, thalassemia, sickle-cell anemia, SCID and
Gaucher's disease.
Methods of gene therapy are well known by those skilled in the art.
Briefly, a sample of CD34-Lin- or CD34-CD38-Lin- cells are isolated from a
source and cultured according to the method of the present invention. The cell
culture is maintained under suitable conditions and the cells are subjected to
techniques for the introduction and stable incorporation of a desired genetic
sequence into the cells. Such introduction techniques may include transfection
(calcium-mediated or microsome-mediated transfection), cell fusion,
electroporation, microinjection or infection using recombinant vaccinia or
retrovirus vectors. The cells which acquire the selected genetic sequence are
then screened for, and reintroduced into a patient. Alternatively, the
identifed
cells may be further cultured to allow the cells to enrich and/or further
differentiate to another cell type prior to being reintroduced into a patient.
The cells of the present invention may be transfected with a selected
DNA sequence encoding for a therapeutic agent such as an antibiotic,
anticancer agents, peptides, cytotoxic compounds and antisense RNA.
Alternatively, the cells may be transfected with an antigenic or immunogenic
product which creates an immune response in a patient and reintroduction. In
this manner, such cells would produce a vaccine like effect.
In a further embodiment of the invention, the cells of the present
invention can be used for fetal genetic testing. The stem cells may be
isolated
from samples of peripheral blood taken from a pregnant woman which contain
some fetal cells. Isolated fetal cells may be cultured and the stem cells
isolated
and tested for genetic abnormalities.
In still a further embodiment of the present invention, the stem cells of
the present invention may be isolated and cultured in vitro and treated with
specific factors and/or cytokines in order to produce a specific cell lineage
such as immune cells, granulocytic, megakaryocytic, etc. which carry out a
2~
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specific function. Such specialized cells may be generated in large numbers
and transplanted back into patients in order to treat them of a disease such
as
for example an autoimmune disease or one of the diseases listed supra.
S Examples
The examples are described for the purposes of illustration and are not
intended to limit the scope of the invention.
Methods of synthetic chemistry, protein and peptide biochemistry and
immunology referred to but not explicitly described in this disclosure and
examples are reported in the scientific literature and are well known to those
skilled in the art.
Example 1 - Analysis of CD34-Lin- cells found in human hematopoietic tissue
Mononuclear cells were isolated from various human hematopoietic cell
sources and stained with monoclonal antibodies for CD2, CD3, CD4, CD7,
CD 13, CD 14, CD 15, CD I 6, CD 19, CD20, and glycophorin conjugated to
FITC, CD38 conjugated to PE and CD34 conjugated to Cy-5 . Cells gated R1
did not express lineage associated markers (Lin-) and were further analyzed
for
the expression of CD34 and CD38.
Identification of CD34- cells with no lineage markers in human hematopoietic
tissues
To determine whether CD34- cells that do not express lineage markers
exist in human hematopoietic tissues, human cord blood cells were first
depleted of mononuclear cells that express 15 different lineage-specific
antigens from human cord blood. This Lin- population was 99% pure (data not
shown). The Lin- cells were then stained with the most widely used CD34 class
III monoclonal antibody conjugated to FITC. Flow cytometric analysis showed
two distinct populations of CD34+ and CD34- cells (Fig lA panel 1). The
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CD34- cells (gated Rl, Fig. lA, I) were collected by flow sorting and
reanalysis
demonstrated their high purity (99%; Fig. 1 A, II). To confirm that these
cells
did not express any surface CD34 antigen, the sorted cells were re-stained
with
two other CD34 Class I and II antibodies that recognize different epitopes of
the CD34 molecule (Fig. lA, III and IV). No CD34+ cells were detected,
attesting to the high purity and lack of CD34 cell surface expression of this
CD34-Lin- population.
To exclude the possibility that CD34 protein was produced in the CD34-
Lin- cells but not transported to the cell surface, cytospins of purified CD34-
Lin- cells were permeabilized, stained with CD34 monoclonal antibodies
conjugated to FITC and counter stained with DAPI (Fig. 1 B). No CD34-FITC
signal was detected in the CD34-Lin- cells. The specificity of the procedure
was shown by the detection of cell surface and intracellular expression of
CD34
on a population of purified CD34+Lin- cells under similar conditions.
Background fluorescence was indicated by staining cells with IgG conjugated
to FITC as isotype control (Fig. 1B). These results indicate that a population
of
Lin- cells exist in human cord blood that does not produce intracellular or
cell
surface CD34.
Heterogeneity within human CD34+Lin- cells is well documented and
further subdivision for the most primitive cells is typically based on the
cell
surface markers CD38, c-kit, Thy-1 and HLA-DR. The expression of these
markers on both the CD34+Lin- and CD34-Lin- cells was compared (Fig. 1 C).
CD34-Lin- cells displayed a bi-modal distribution of CD38, clearly dividing
the
population into two fractions in contrast to the high proportion of CD34+Lin-
cells that express CD38 (Fig. 1C). Cell surface expression of c-kit was
similar
between that two populations, while the CD34-Lin- cells are almost exclusively
Thy-1- and HLA-DR- (Fig. IC). Both the absence of HLA-DR expression and
the presence of Thy-1 have been proposed to define more primitive
subfractions within the CD34+Lin- population. Therefore, the CD34-Lin-
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population derived from cord blood is a distinct population which differs not
only in CD34 expression from primitive CD34+Lin- cells but also in
phenotypic heterogeneity based on additional markers associated with stem
cells.
Example 2- Cell Immunostainine
Cord blood cells purified by flow cytometry as done previously ( 11, 12)
were cytospun onto slides, permeabilized, and incubated in a BSA solution. The
results are shown in Figure 1B. (Column I) CD34+CD38-Lin- cells were
stained with isotype control antibody conjugated to FITC (Becton Dickinson)
and countered stained with DNA binding DAPI as a control for non-specific
background flourescence. (Column II) CD34+CD38-Lin- cells and (Column
III) CD34-Lin- cells were stained with CD34 monoclonal antibodies followed
by DAPI counter stain. All slides were examined using a fluorescent
1 S microscope utilizing the appropriate filters for DAPI to detect the
nucleus of
cells and FITC for the presence of CD34 protein.
Both CD34-Lin- and CD34+Lin- purified cells were.stained with
monoclonal antibodies conjugated to fluorochromes for CD38 (Becton
Dickinson, BD), c-kit (BD), Thy-1 (Coulter) and HLA-DR (BD). Stained
populations were then washed and analyzed using standard flow cytometric
techniques (J. Exp Med, PNAS) followed by the display of histograms using
the Cell Quest software program (BD) (n=3).
Example 3 - Cell Engraftment
Purified cell populations at the indicated dose were transplanted by tail
vein injection into sublethally irradiated mice (375 cGy using a 137Cs g-
irradiator) according to a standard protocol as previously described (16, 17).
Mice were sacrificed 8 to 12 weeks post transplant and the bone marrow from
the femurs, tibiae and iliac crests of each mouse were flushed into IMDM
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containing 10% FCS. Mouse bone marrow was analyzed using FACS analysis
and by southern analysis using genomic DNA extracted by standard protocols
in which the level of human cell engraftment was determined by comparing the
characteristic 2.7 kb band with those of human:mouse DNA mixtures as
controls (limit of detection 0.05% human DNA) ( 16, 17). The results are
shown in Figures 2 and 7.
Example 4 - Determination of Hematopoietic Progenitor Activity of CD34-Lin,
CD34+CD38-Lin- and CD34-CD38+Lin-
Highly purified cells were plated in clonogenic methlycellulose assays
and seeded on MS-5 stroma in order to quantitate the CFC and LTC-IC content,
respectively. Clonogenic capacity of CD34-Lin- cells was extremely low in
comparison to CD34+CD38-Lin- cells (250 CFC vs. 8.9 CFC per 800 cells)
(Table I). As many as 10,000 cells needed to be seeded on MS-5 stroma to
detect a single LTC-IC within the CD34-Lin- cell fraction, while further
purification demonstrated that detection of LTC-IC in the CD34-CD38-Lin-
fraction required seeding of at least 2000 cells. By contrast, as few as 10
CD34+CD38-Lin- cells contain an LTC-IC. The CD34-CD38+Lin- cells were
devoid of LTC-IC activity (limit of detection at 10,000 cells) but contained a
much higher capacity to form CFC specifically committed to the erythroid
lineage (Table I). The low efficiency of production of myeloid and erythroid
committed progenitors, as well as the more primitive LTC-IC, is similar to
observations made with murine CD34-Lin- cells in the same assay systems and
suggest that functional similarities may exist between the CD34-Lin- cells
from
these two species (8, 10).
Example 5 - Multilinea~e Differentiation of Human CD34-Lin- cells in
NOD/SCID mice after ex vivo Culture
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A representative mouse was transplanted with 50,000 expanded CD34-
Lin- cord blood cells after 2 days of ex-vivo culture in the presence of SF
medium supplemented with 5% FCS. Mouse bone marrow was extracted 10
weeks after transplant and analyzed by multiparameter flow cytometry (11, 12).
The results are shown in Figure 8.
CD34- Cell Culture with Growth Factors
CD34-Lin- cells were incubated in 50 ml of SF condition consisting of
IMDM supplemented with 1% BSA (Stem Cell Technologies), ~ mg/ml of
human insulin (Humulin R from Eli Lilly and Co.), 100 mg/ml of human
transferrin (Gibco, BRL), 10 mg/ml of low density lipoproteins (Sigma
Chemical Co.), 10-4 M Beta-mercaptoethanol and growth factors (GF). GF
cocktail was used at final concentrations of 300 ng/ml of SCF (Amgen) and Flt-
3 (Immunex), 50 ng/ml of G-CSF (Amgen), 10 ng/ml of IL-3 (Amgen) and IL-
6 (Amgen). 25% of condition media obtained from a fresh umbilical vein
endothelial cell culture in a low percentage of serum ( 10%) and passaged four
times, was added in some wells. Cells were cultured in flat bottomed
suspension wells of 96-well plates (Nunc), incubated for 2 and 4 days at 37oC
and 5% C02 and SO ml of fresh GF cocktail was added to each well every other
day.
Example 6 - Effect of ex vivo culture on the number of clonogenic progenitors
present in the CD34-Lin-, CD34-CD38-Lin- and CD34-CD38+Lin- cell
fractions
An aliquot of 800 to 2, 500 CD34-Lin-, CD34-CD38-Lin- or CD34-
CD38+Lin- cells were plated in clonogenic progenitor assays under standard
conditions at the initiation of ex vivo cultures (day 0). Cells present after
4 days
of culture in the presence of SF or SF supplemented with 5% FCS or 2~% of
HLJVEC-CM were plated in the same conditions. The number of CFC/800
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input cells were estimated (mean ; SEM ; n=number of experiment). The
results are seen in Table II.
Example 7 - The effect of liquid culture on the development and potential
differentiation of CD34-CD38-Lin- cells
Individual wells were analyzed by flow cytometry after 2 and 4 days of
culture in various conditions (Fig. 6). CD34-CD38-Lin- cells seeded in SF
media began to express CD34 which could be enhanced with the addition of
serum (Fig. 6). In contrast, the majority of cells obtained after 2 or 4 days
of
culture in the presence of 25% HUVEC (human umbilical vein endothelial cell)
conditioned medium still maintained the same phenotype. The acquisition of
CD34 demonstrates the differentiation capacity of CD34-CD38-Lin- cells in-
vitro. The production of CD34+CD38- cells suggests that CD34-CD38-Lin-
cells precede CD34+CD38- population in the hierarchy of human
1 ~ hematopoiesis.
Example 8- Cytokine stimulation of CD34-CD38-Lin- Repopulating Activity
To evaluate the repopulating activity of CD34-Lin- and more highly
purified CD34-CD38-Lin- cell fractions after ex vivo culture, cultured cells
were injected into NOD/SCID mice and the level of human engraftment
evaluated after 8 to 10 weeks. Purified cell fractions from 44 CB and 3 BM
samples cultured for 2 and 4 days in SF, SF supplemented with 5% FCS or 25%
HUVEC-CM were transplanted at various doses into 144 recipient NOD/SCID
mice and the level of human cell engraftment was determined (Figure 7B). A
total of 15 of 35 mice were engrafted following transplantation with CD34-Lin-
cells that had been cultured for 4 days in SF or 5% FCS at cell doses below
the
calculated frequency of CD34-SRC. For example, 13 of 29 mice were
engrafted following transplantation of 50,000 to as few as 4,000 cultured
CD34-Lin- cells. By contrast, only 1 of 7 mice were engrafted when 100,000
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uncultured CD34-Lin- cells were transplanted (Figure 2). Similarly. a higher
proportion of mice (33%) transplanted with cultured CD34-CD38-Lin- cells
were engrafted (Figure 7B) compared to mice transplanted with similar doses
of uncultured CD34-CD38-Lin- (Figure 7B). Quantitative analysis using
Poisson statistics indicated a frequency of 1 CD-SRC in 38,000 cultured CD34-
CD38-Lin- cells (range 1/22,000 to 1/71.000). It was not possible to calculate
the frequency of CD34-SRC in uncultured CD34-CD38-Lin- cells (Figure 7A)
because there was infrequent engraftment despite injection of as many as
50.000 cells. This result indicates that the actual stem cell frequency of
freshly
isolated cells must be much lower compared to cultured cells. The human
lineage distribution in mice transplanted with expanded CD34-Lin- and CD34-
CD38-Lin- cells is similar to that with unstimulated purified CD34-Lin- cells
(Figure 8). In addition, the inability of cultured CD34-CD38+Lin- cells to
engraft mice confirms the absense of repopulating cells within this fraction.
It
was evident that culture conditions, especially HLJVEC-CM, induce an increase
in the number of CD34-SRC in the absence of cell proliferation.
The bone marrow of engrafted mice was analyzed by multiparameter
flow cytometry to determine whether cultured CD34-Lin- repopulating cells
possessed the same in vivo proliferative and differentiative capacity as
uncultured cells. A representative analysis of the bone marrow of a NOD/SCID
mouse transplanted with an initial population of 40,000 CD34-Lin- cells after
4
days of culture is shown in Figure 8. The bone marrow of this mouse contained
7% human cells as detected by expression of CD45, a human specific pan-
leukocyte marker (Fig. 8). Both B and T-lymphoid cells were present in the
murine bone marrow as shown by staining for CD 19, CD20 and CD4, CD3
antigens (Fig. 8D-F). The presence of CD33+, CD14+, CD15+ and CD13+
cells indicated the differentiation potential of CD38-CD38-Lin- cells to the
myeloid lineages (Fig. 8G-H). The engraftment pattern of mice transplanted
with expanded CD34-CD38-Lin- cells is similar to that observed with
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WO 99/23205 PCT/CA98/01012
unstimulated purified CD34-Lin- cells. The presence of human T-cells is a
unique feature of CD34-Lin- engraftment, since T-cells have not been detected
in mice transplanted with purified CD34+CD38-Lin- cells either before or after
ex vivo culture.
S
Examt~le 9 - Testing of Whether CD34-SRC are Biolo~icallv Distinct from
CD34+SRC.
To test whether the CD34+SRC are lost if CD34+CD38-Lin- or
CD34+Lin- cells are cultured in the presence of serum, CD34+CD38-Lin- and
CD34-Lin- cells from the same human CB sample were cultured for 4 days
under the dame serum containing conditions. In a representative experiment, 3
out of 6 mice were engrafted following transplantation with cultured CD34-
Lin- cells (Figure 9). In contrast, 5,000 and 10,000 CD34+CD38-Lin- cells,
containing 10-20 CD34+SRC, cultured under the same conditions were unable
to engraft NOD/SCID mice. These results provide independent confirmation
that the repopulating cells derived from the CD34-Lin- subfraction are
biologically distinct from those derived from CD34+CD38-Lin- cells.
For the DNA analysis shown in Figure 9, NOD/SCID mice were
transplanted with CD34-Lin- or CD34+CD38-Lin- cells purified from the same
cord blood sample and cultured in the presence of 5% FCS. A southern blot
was performed using standard techniques and was hybridized with a human
chromosome 17-specific a-satellite probe.
The present invention is not limited to the features of the embodiments
described herein, but includes all variations and modifications within the
scope
of the claims.
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Table 1
Hematopoietic Progenitor Activity of CD34-Lin- CD34-CD38-Lin
and CD34-CD38+Lin
Phenotype of purified CFC/800 cells Frequency of
population LTC-IC
CD34-Lin- 8.9~3 <1/10,000 (n=2) exp #1
(n=4) 5,501~1640(n=2) exp#2
CD34-Lin-CD3 8- 2.7~2 < 1 /2,000
(n=36) (n=4)
CD34-Lin-CD38+ 77~31 <1/10,000 (n=10)
(n=4)
CD34+CD38- 250~46 >1/10
(n=4) (n=4)
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Table 2
Effect of ex vivo culture on the number of clonogenic progenitors
present in the CD34-Lin-, CD34-CD38-Lin- and CD34-CD38+Lin- cell
fractions.
Phenotype of purified Day Medium Number [?]
~o~ulation
CD34-Lin- 8.9 3 Serum Free 10 0.9 (n=4)
(n=4) 5% Serum 76 21 (n=2)
CD34-Lin-CD38- 2.7 2 5% Serum 6.4 4.~ (n=7)
(n=36) Serum Free 1.2 1 (n=9)
25% HUVEC0.5%
0.5 (n=7)
CD34-Lin-CD38+ 77 31 Serum Free 20 10 (n=4)
(n=10) 5% Serum 134 41 (n=4)
*CFC/800 Input Cells .
3~
