U.S. patent application number 10/628886 was filed with the patent office on 2011-04-21 for human brain endothelial cells and growth medium and method for expansion of primitive cd34+cd38- bone marrow stem cells.
Invention is credited to Dennis J. Chute, John P. Chute, Thomas A. Davis, Abba A. Saini.
Application Number | 20110091426 10/628886 |
Document ID | / |
Family ID | 22341823 |
Filed Date | 2011-04-21 |
United States Patent
Application |
20110091426 |
Kind Code |
A1 |
Chute; John P. ; et
al. |
April 21, 2011 |
Human brain endothelial cells and growth medium and method for
expansion of primitive CD34+CD38- bone marrow stem cells
Abstract
A novel co-culture system using human brain endothelial cells
(HUBEC) which promotes the expansion of human CD34.sup.+CD38.sup.-
cells consistent with the PMVEC system is disclosed. HUBEC were
isolated from cadaveric donors, passed in primary culture, cloned
and found to be Von Willebrand Factor positive. Cultivation of
purified bone marrow CD34.sup.+ cells on HUBEC monolayers
supplemented with GM-CSF+IL-3+IL-6+SCF+flt-3 ligand caused a
14.5-fold increase in total cells, an 6.6-fold increase in
CD34.sup.+ cells, and, most remarkably, a 440-fold increase in
CD34.sup.+CD38.sup.- cells after 7 days. Further, CFU-GM production
increased 15.1-fold, BFU-E increased 8-fold, and CFU-Mix increased
5.2-fold. Optimal generation was dependent upon the continued
presence of exogenous supplied cytokines. In comparison,
identically treated stroma-free suspension cultures supported a
10.2-fold expansion of total cells, a 3-fold increase in CD34.sup.+
cells and maintained the CD34.sup.+CD38.sup.- cell pool after 7
days of culture. Moreover, we found that non-brain human
endothelial cells isolated from the same donors supported neither
the expansion nor the maintenance of human CD34.sup.+CD38.sup.-
cells. Although few steady state CD34.sup.+CD38.sup.- cells give
rise to visible colony-forming cells in methylcellulose cultures,
our FACS based cell cycle and sorting experiments demonstrated the
activation of a highly clonogenic CD34.sup.+CD38.sup.- population
(24% cloning efficiency) during ex-vivo culture on cytokine treated
HUBEC. These results suggest that bone marrow CD34.sup.+CD38.sup.-
cells require a stromal cell microenvironment for optimal expansion
and that ex-vivo expanded CD34.sup.+CD38.sup.- cells generated in
the HUBEC culture system appear to retain some degree of primitive
"stemness".
Inventors: |
Chute; John P.; (Bethesda,
MD) ; Saini; Abba A.; (Bethesda, MD) ; Chute;
Dennis J.; (Baltimore, MD) ; Davis; Thomas A.;
(Newton, PA) |
Family ID: |
22341823 |
Appl. No.: |
10/628886 |
Filed: |
July 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09452855 |
Dec 3, 1999 |
6642049 |
|
|
10628886 |
|
|
|
|
60112042 |
Dec 4, 1998 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61P 43/00 20180101;
C12N 2501/22 20130101; A61K 2035/124 20130101; C12N 2501/23
20130101; C12N 2501/26 20130101; C12N 5/0647 20130101; A61P 7/00
20180101; C12N 2501/125 20130101; C12N 2502/28 20130101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61P 43/00 20060101 A61P043/00 |
Claims
1-8. (canceled)
9. A method of engrafting human bone marrow CD34+CD38-
hematopoietic progenitor cells in a human, the method comprising
the steps of: i) contacting the isolated CD34+CD38- hematopoietic
progenitor cells with human brain endothelial cells containing a
factor or factors that expand the CD34+CD38- hematopoietic
progenitor cells; ii) co-culturing the contacted CD34+CD38-
hematopoietic progenitor cells and endothelial cells in the
presence of at least one cytokine in an amount sufficient to
support amplification/expansion of said CD34+CD38- hematopoietic
progenitor cells; iii) isolating the amplified/expanded CD34+CD38-
hematopoietic progenitor cells from the culture; and iv) infusing
the amplified/expanded CD34+CD38- hematopoietic progenitor cells
into said human.
10. A method of engrafting human bone marrow CD34+CD38-
hematopoietic progenitor cells in a human, the method comprising
the steps of: i) isolating CD34+CD38- hematopoietic progenitor
cells from human bone marrow; ii) contacting the isolated
CD34+CD38- hematopoietic progenitor cells with human brain
endothelial cells containing a factor or factors that expand the
CD34+CD38- hematopoietic progenitor cells; iii) co-culturing the
contacted CD34+CD38- hematopoietic progenitor cells and endothelial
cells in the presence of at least one cytokine in an amount
sufficient to support amplification/expansion of said CD34+CD38-
hematopoietic progenitor cells; iv) isolating the
amplified/expanded CD34+CD38- hematopoietic progenitor cells from
the culture; and v) infusing the amplified/expanded CD34+CD38-
hematopoietic progenitor cells into said human.
11. The method according to claim 9 or 10, wherein said cells are
isolated from the bone marrow of the human.
12. The method according to claim 9 or 10, wherein said cells are
isolated from the bone marrow of the human in need of said
CD34+CD38- hematopoietic progenitor cells.
13. The method according to claim 9 or 10, wherein said CD34+CD38-
hematopoietic progenitor cells are isolated from the bone marrow of
a donor.
14-25. (canceled)
Description
RELATED APPLICATION
[0001] Benefit of U.S. Provisional Application 60/112,042 filed 4
Dec. 1998, which is incorporated herein by reference, is
claimed.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to a growth medium derived from human
brain endothelial cells (HUBEC) and the methods of utilizing said
growth medium to expand bone marrow stem cells.
[0004] 2. Description of the Prior Art
[0005] The development of an ex-vivo system which supports the
proliferation and expansion of the most primitive hematopoietic
stem cells (HSC) would have direct application to the fields of
gene therapy and stem cell transplantation. Identification and
characterization of the optimal culture conditions for the
expansion of long-term repopulating cells is a requirement for gene
therapy protocols and other stem cell-based therapies.
[0006] Various cytokine combinations and liquid culture methods
have been shown to support the proliferation of CD34.sup.+ HPC in
vitro, but the most primitive CD34.sup.+CD38.sup.- cells are
frequently lost due to differentiation and cell death [1-6]. In
contrast, other investigations have demonstrated that when human
HPC are co-cultured in contact with autologous, allogeneic, and
xenogeneic bone marrow stroma, a small percentage of long term
culture initiating cells (LTC-IC) can be maintained over several
weeks [7-9]. Similarly, others have reported the expansion and
differentiation of LTC-IC and CFC in stroma-free liquid suspension
cultures using exogenous cytokines plus conditioned medium from
bone marrow stromal cultures [10-12]. Most recently, it was
reported that human cord blood CD34.sup.+ cells could be maintained
in stroma-free liquid cultures in the presence of flt-3 ligand,
megakaryocyte growth and development factor (MGDF), SCF, and IL-6
for up to 10 weeks without losing their ability to repopulate
NOD/SCID mice [13].
[0007] Vascular endothelium, reticuloendothelial elements, and
hematopoietic cells of all types have been postulated to arise from
hemangioblasts, a primitive embryonic cell of mesodermal origin
[14,15]. During the earliest stages (day 7-8 postcoitum) of
mammalian embryonic hematopoiesis, primitive hematopoietic stem
cells are found encased in blood islands which derive from
aggregates of mesodermal cells which have colonized the embryonic
yolk sac [16]. Bone marrow, umbilical vein, and murine yolk sac
endothelial cell lines have been shown to elaborate a number of
growth factors that regulate early hematopoiesis [17-20]. In
addition, the long term proliferation and differentiation of
myeloid, erythroid, and megakaryocytic progenitor cells has been
demonstrated in vitro using microvascular endothelial cells derived
from adult bone marrow and embryonic yolk sac [18,19]. However, the
fate of the most primitive CD34.sup.+CD38.sup.- progenitor cells
following co-culture with endothelial cell monolayers has not been
well demonstrated. Previously, we reported that a primary porcine
microvascular endothelial cell line (PMVEC) supports a rapid and
robust expansion of human hematopoietic cells exhibiting the
primitive CD34.sup.+CD38.sup.- phenotype [21,22]. Unlike other
reported co-culture systems, we have demonstrated that
CD34.sup.+CD38.sup.- cells expanded on brain endothelium retain the
ability to successfully engraft in vivo in both a SCID-Hu bone
model [23] and in lethally irradiated baboons [24].
[0008] Human brain vascular endothelial cells are similar to other
sources of endothelial cells in that they develop cobblestone
morphology in-vitro [25], and they express cell adhesion molecules
(selectins, integrins) which mediate the "rolling", adherence, and
trafficking of leukocyte [26,27]. Based upon our observations of
the hematopoietic capacity of PMVEC and recognizing the limitations
of applying a porcine endothelial cell line in human clinical
studies, we isolated primary human brain endothelial cells (HUBEC)
and evaluated their capacity to support the ex-vivo expansion of
human CD34.sup.+CD38.sup.- cells. Our results indicate that human
brain endothelial cells support a unique expansion and apparent
self-renewal of the most primitive CD34.sup.+CD38.sup.- HPC at a
level comparable to our observations with porcine endothelial
cells. Further investigations evaluating the in vivo repopulating
potential of HUBEC-expanded HPC will be important in implementing
future gene therapy, cord blood expansion, and stem cell transplant
protocols.
SUMMARY OF THE INVENTION
[0009] Accordingly, an object of this invention is a growth medium
based on human brain endothelial cells (HUBEC).
[0010] Another object of the invention is the growth factor
contained within the medium that is elaborated by the HUBEC and
promotes the expansion of primitive CD34+CD38- bone marrow stem
cells.
[0011] A further object of this invention is a method for expanding
the population of primitive CD34+CD38- bone marrow stem cells.
[0012] Yet another object of this invention is the treated,
concentrated product of the growth medium containing the growth
factor.
[0013] An additional object of the invention is a growth medium
that can be used for GMP production of expanded cells.
[0014] These and additional objects of the invention are
accomplished by human brain endothelial cells (HUBEC) that can
serve as a uniquely supportive hematopoietic microenvironment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] A more complete appreciation of the invention will be
readily obtained by reference to the following Description of the
Preferred Embodiments and the accompanying drawings in which like
numerals in different figures represent the same structures or
elements. The representations in each of the figures are
diagrammatic and no attempt is made to indicate actual scales or
precise ratios. Proportional relationships are shown as
approximations.
[0016] FIG. 1. Phenotype of HUBEC in primary culture. A. typical
cobblestone morphology of HUBEC (passage 10) from a confluent
(40.times. magnification). B. Von Willebrand expression by cultured
HUBEC (passage 10) was analyzed by flow cytometeric analysis.
Isotype-matched control Ab is indicated, by a heavy solid line
while FITC-conjugated anti-human Von Willebrand staining is
depicted by the dotted line.
[0017] FIG. 2. Morphology of a typical adherent colony of
hematopoietic cells following 7 days of co-culture of human bone
marrow CD34+ cells on HUBEC monolayers treated with Granulocyte
monocyte colony stimulating factor (GMCSF)+Interleukin-3
(IL-3)+Interleukin-6 (IL-6)+Stem cell factor (SCF)+fetal liver
tyrosine kinase-3 ligand (flt-3 ligand). [0018] A) Dispersed colony
of hematopoietic cells adherent to HUBEC monolayers after vigorous
washing (40.times.). [0019] B) Adherent colony of hematopoietic
cells on HUBEC monolayer stained with Wrights' Geimsa stain
(100.times.).
[0020] FIG. 3. Flow cytometric analysis of expanded CD34+ bone
marrow cells following HUBEC co-culture vs. stroma-free liquid
culture vs. Human non-brain endothelial cell co-culture. Purified
human CD34+ cells were seeded on HUBEC monolayers or in stroma-free
liquid culture or in co-culture with non-brain endothelial cell
monolayers in the presence of GMCSF+IL-3+IL-6+SCF+flt-3 ligand and
cultured for 7 days. The phenotype of purified bone marrow CD34+
cells at day 0 (input) is shown in (A). After 7 days, non-adherent
hematopoietic cells were harvested from the HUBEC co-cultures (B),
the liquid suspension cultures (C), and the non-brain endothelial
cell co-cultures (D), and stained with FITC-conjugated CD34 MoAb
and PE-conjugated CD38 MoAb and analyzed by flow cytometry. Log
fluorescence distribution of CD34 expression is shown along the
X-axis and CD38 expression along the Y-axis. Each result is shown
with its isotype control.
[0021] FIG. 4. Morphology of the CD34+CD38- cells following 7 days
of HUBEC co-culture. Following 7 days of co-culture of human CD34+
cells on HUBEC monolayers treated with GMCSF+IL-3+IL-6+SCF+flt-3
ligand, the non-adherent cells were harvested and stained with
FITC-conjugated CD34 and PE-conjugated CD38. Representative
CD34+CD38- cells collected by Fluorescence activated cell sorting
(FACS) and stained with Wrights' Geimsa are shown at
100.times..
[0022] FIG. 5. Cell cycle status of bone marrow CD34+CD38- cells at
day 0 and following 7 days of HUBEC co-culture. Bone marrow CD34+
cells were stained with CD34APC, CD38PE, Ki67FITC, and 7AAD to
assess for cell cycle status. [0023] In (A), bone marrow CD34+CD38-
cells are shown based upon their staining for Ki67FITC and 7AAD.
[0024] In (B), CD34+CD38- cells are shown using the same
stains.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] Bone marrow CD34.sup.+CD38.sup.- cells are highly enriched
for pluripotent progenitor cells which account for long term
repopulation in vivo [31-33], but attempts at expanding
CD34.sup.+CD38.sup.- cells in-vitro for therapeutic use have had
very limited success due to the differentiation and cell death
which frequently occurs when these primitive cells are exposed to
cytokines [3,6,34]. An ex-vivo co-culture system which has the
capacity to expand the population of long term repopulating cells
while maintaining their CD34.sup.+CD38.sup.- phenotype would have
immediate clinical applications in gene therapy, cord blood
expansion, and stem cell transplantation protocols.
[0026] In this study, we demonstrate that primitive hematopoietic
progenitor/stem cells actively proliferate and expand in direct
association with preformed HUBEC monolayers, which is consistent
with our previous observations using a porcine endothelial cell
line [21]. Unlike liquid suspension cultures and non-CNS derived
endothelial cell cultures, co-culture with HUBEC is essential for
expansion of the primitive CD34.sup.+CD38.sup.- subset (440-fold at
day 7) while maintaining their primitive phenotype and immature
undifferentiated blast cell morphology. In addition, CD34.sup.+
cell proliferation in HUBEC co-culture appears to be the greatest
in the CD34.sup.+CD38.sup.- cell population. While the addition of
exogenous growth factors including GM-CSF, IL-3, IL-6, SCF and
flt3-ligand are important for CD34.sup.+ cell activation and
expansion, additional as-yet unidentified endothelial cell factors
most likely play a critical role in the CD34.sup.+CD38.sup.- cell
"self-renewal" processes [35]. In contrast, results from
stroma-free and non-brain endothelial cell cultures demonstrate
that cultured CD34.sup.+CD38.sup.- fail to proliferate
significantly, differentiate quickly, and overall CFC cell
expansion is limited and short lived. These results suggest that
brain-derived endothelial cells provide a unique microenvironment
which promotes the cell division and apparent self-renewal of the
primitive CD34.sup.+CD38.sup.- population.
[0027] We confirm that the majority of steady state bone marrow
CD34.sup.+CD38.sup.- cells are quiescent and reside primarily in
G.sub.0 of the cell cycle [22,36,37]. The lack of cell cycling
induction within the most primitive CD34.sup.+CD38.sup.- population
has been identified as a major impediment to the successful
transduction of these cells with retroviral based gene vectors
[36,38]. We have previously determined that CD34.sup.+CD38.sup.-
cells are easily recruited into cell cycle when cultured on porcine
brain endothelial cells (PMVEC) [22]. In the current study, we
found that primary HUBEC cultures from numerous donors in
combination with exogenous cytokines induced the majority (>70%)
of the previously quiescent CD34.sup.+CD38.sup.- population to
enter G.sub.1 or G.sub.2/S/M phase of cell cycle after 7 days.
Although the mechanism of rapid expansion of CD34.sup.+CD38.sup.-
cells is unclear, HUBEC may provide the microenvironment necessary
in combination with exogenous cytokines to induce rapid cycling and
preserve the "sternness" of very primitive HPC (<2% of the total
CD34.sup.+ cells used to initiate cultures) and may also prevent
apoptotic cell death. In contrast, we have found that this high
level of cell division in CD34.sup.+CD38.sup.- cells is not
achievable in stroma-free liquid and human non-brain endothelial
cocultures supplemented with GMCSF+IL-3+IL-6+SCF+flt-3 ligand.
Unlike other stromal based culture systems [10,11], we do not
observe inhibitory effects of endothelial cell contact on
CD34.sup.+CD38.sup.- expansion. In fact, in the HUBEC system,
cell-to-cell contact promotes maximal expansion of the
CD34.sup.+CD38.sup.- cell population which is dependent upon the
addition of a combination of exogenous growth factors and appears
to override any type of direct endothelial cell inhibitory effects.
These findings are consistent with our previously reported
observations that CD34.sup.+CD38.sup.- cell expansion is optimal
when CD34.sup.+ cells are cultured directly in contact with PMVEC
monolayers rather than when cultured separately from the
endothelial feeder cells using transwell inserts [21]. Since HUBEC
provide a microenvironment which supports a high level of cell
cycling and expands the primitive CD34.sup.+CD38.sup.- population,
this culture system may also promote higher efficiencies of gene
transfer into transplantable cells using standard retroviral
vectors.
[0028] Bone marrow CD34.sup.+CD38.sup.- cells contain long term
culture initiating cells (LTC-IC) which give rise to CFC over 6
weeks when cultured with stromal feeder layers [29,39]. Our
clonogenic data in this study is consistent with that reported by
others demonstrating that steady state CD34.sup.+CD38.sup.- cells
do not give rise to significant numbers of CFC when cultured
directly in 14 day methylcellulose cultures plus cytokines [29,39].
For that reason, CD34.sup.+CD38.sup.- cells typically have been
characterized as having limited CFC activity. It has previously
been reported that 10 day serum-free liquid cultures of human
CD34.sup.+CD38.sup.- cells with optimal cytokine combinations
including flt-3 ligand, SCF, and IL-3 promoted a 30-fold increase
in LTC-IC production by the expanded population [30]. Although the
authors did not address the phenotype of the expanded population in
that study, it is likely that the majority of the input
CD34.sup.+CD38.sup.- cells exposed to cytokines for 10 days
underwent significant differentiation and lineage commitment. In
contrast, we demonstrate in this study that co-culture of human
CD34.sup.+CD38.sup.- cells with preformed HUBEC monolayers plus
cytokines supports rapid cycling and ex vivo expansion of
phenotypically primitive HPC of the CD34.sup.+CD38.sup.- subset.
Since the HUBEC co-culture supports this pronounced increase in
CD34.sup.+CD38.sup.- cells, we were easily able to collect and
study this rare subset of long term repopulating cells to
interrogate their biology after 7 days of co-culture. Unlike
CD34.sup.+CD38.sup.- cells in the steady state,
CD34.sup.+CD38.sup.- cells expanded on HUBEC monolayers directly
give rise to hundreds of colonies of myeloid, erythroid, and mixed
lineages in methylcellulose at a cloning efficiency of 24%. This
suggests a period of pre-incubation in HUBEC co-culture plus
cytokines can stimulate early HPC (stromal cell responsive
progenitor cells) which would normally be cytokine unresponsive in
a stroma-free microenvironment. In previous studies, we have shown
that HSC expanded in PMVEC coculture are capable of competitive
myeloid and lymphoid marrow repopulating when implanted into
SCID-hu-bone and transplanted into lethally irradiated baboons
[23,24]. Together these findings demonstrate the requirement for
direct stem cell-stromal cell interaction in order to optimize HPC
survival, expansion, and maintenance of HPC function under ex-vivo
culture conditions and to preserve graft quality. Moreover, the
ability to determine stroma cell dependent CFC frequencies in a
short time interval makes the HUBEC culture system an attractive
alternative to other long-term in vitro quantification
methodologies. Likewise, the ability to activate and significantly
expand CD34.sup.+CD38.sup.- progenitor cell pool has potential
ramifications in clinical stem cell expansion studies.
[0029] Recently, bone marrow microvascular and human umbilical vein
endothelial lines have been used to support the short-term growth
and proliferation of human CD34.sup.+ progenitor cells [18,20].
However, the outcome of the primitive CD34.sup.+CD38.sup.-
subpopulation has not been detailed in these co-culture systems.
More recently, a stromal cell line derived from murine fetal liver,
AFT024, has been shown to support the maintenance of a small
percentage of CD34.sup.+CD38.sup.- cells over 3-10 days of
co-culture [40]. The authors hypothesized in this study that the
AFT024 cell line may have maintained extended long term culture
initiating cells (ELTC-ICs) by inhibiting cell cycling and
differentiation of these CD34.sup.+CD38.sup.- cells [40]. In
contrast to these observations, HUBEC co-culture induces a high
level of cell cycling in the quiescent CD34.sup.+CD38.sup.- subset
and the absolute percentage of CD34.sup.+CD38.sup.- cells is not
only maintained, but increases .about.440-fold (0.3% at day 0 to
10.5%) at day 7. These data suggest that human brain endothelial
cells may provide other hematopoietic signal(s) such as soluble
growth factors, membrane-bound growth factors, extracellular matrix
proteins, or cellular adhesion molecules, which are unique from
fetal liver, bone marrow, or umbilical vein endothelial cell
lines.
[0030] Since human brain endothelial cells support the apparent
self renewal and expansion of primitive HPC whereas non-brain
endothelial cells from the same donor do not, we speculate that the
biology of brain endothelial cells may be similar to embryonic and
extra-embryonic endothelial cells which are critically involved in
the generation of hematopoietic stem cells during embryogenesis
[14-16]. Recently, it was reported that a murine
aorto-gonad-mesonephros (AGM) region derived endothelial cell line
(DAS 104-4) was capable of maintaining a small fraction of murine
CD34.sup.+ Sca-1.sup.+ c-kit.sup.+ lin.sup.- cells over 7 days of
co-culture and these hematopoietic cells retained their in vivo
reconstituting capacity [41]. Based upon their findings, the
authors hypothesized that the DAS 104-4 AGM-derived cell line was
able to support the self renewal of a small percentage of
hematopoietic stem cells [41]. Based upon their similar capacities
to maintain primitive hematopoietic progenitor cells ex vivo, it is
plausible that human brain endothelial cells may possess similar
hematopoietic properties to AGM-derived endothelial cells. In
addition, the profound induction of cell cycling and expansion of
the CD34.sup.+CD38.sup.- subpopulation observed on HUBEC monolayers
suggests that brain endothelial cells may be providing novel
hematopoietic signals as well.
[0031] In comparison to other ex-vivo cultures systems, we believe
that the human brain endothelial cell (HUBEC) culture system has
several major advantages which will prove useful in future clinical
stem cell expansion and gene therapy studies. First, rapid
amplification and collection of very large numbers cycling
CD34.sup.+CD38.sup.- cells can occur within 1 week of culture.
Second, expansion of hematopoietic progenitor cells requires only
preformed endothelial monolayers, which are easy to establish and
maintain, plus a defined combination of commercially available
cytokines. Third, we have shown that expansion of
CD34.sup.+CD38.sup.- cells requires only human brain endothelial
cells (single cell type) whose hematopoietic biology should be more
easily dissected compared to heterogeneous stromal cell systems.
Fourth, we have previously shown that HPCs expanded on porcine
brain endothelial cell monolayers retain both in vivo myeloid and
lymphoid repopulating potential with no apparent engraftment
defects [23,24]. Results from ongoing SCID-Hu and primate bone
marrow transplantation studies utilizing primitive
CD34.sup.+CD38.sup.- cells expanded on HUBEC monolayers will be
important in evaluating this system for future therapeutic
applications.
DEFINITIONS
[0032] 1. Human CD34+ hematopoietic stem cells (HSC): CD34+ cells
isolated from various hematopoietic tissues (not limited to
peripheral blood, bone marrow, cord blood, spleen, and liver) that
are capable of full and permanent lymphoid, erythroid and myeloid
reconstitution following transplantation into a lethally irradiated
recipient. These cells are a subset of the CD34+CD38- HPC
population. [0033] 2. HPC: Hematopoietic progenitor cells [0034] 3.
CD34+CD38+ hematopoietic progenitor cells: Committed/differentiated
CD34+ hematopoietic progenitor cells that express the lineage
commitment surface marker CD38 and are functionally described as
having only short-term hematopoietic reconstitution in vivo. [0035]
4. CD34+CD38- hematopoietic progenitor cells: The undifferentiated
subset of CD34+ HPC cells that lacks CD38 expression and contains
hematopoietic stem cells (HSC) which are functionally capable of
long-term hematopoietic reconstitution. The HSC are located in the
population of cells [0036] 5. SCF: Stem cell factor [0037] 6. IL-6:
Interleukin-6 [0038] 7. MGDF: Megakaryocyte growth and
developmental factor [0039] 8. Long-term culture-initiating cells
(LTC-IC): HSC that are defined by their potential to grow,
proliferate and be maintained in stroma based cultures systems in
the absence of exogenous cytokines over 5-7 weeks of culture.
[0040] 9. Colony-forming cells (CFC): Committed progenitor cells
(CD34+CD38+) that give rise to assayable in vitro colonies of
either the myeloid, erythroid, or lymphoid lineages following 14
days of culture. [0041] 10. HUBEC: Human brain derived endothelial
cells [0042] 11. GM-CSF: granulocyte macrophage colony-stimulating
factor. [0043] 12. IL-3: Interleukin-3 [0044] 13. MoAb: Monoclonal
antibody [0045] 14. FACS: Fluorescent activated cell sorting [0046]
15. PE-CD38: Phycoerythrin conjugated anti-CD38 antibody [0047] 16.
FITC-CD34: Fluorescein Isothiocyanate anti-CD34 antibody [0048] 17.
SID: Surface intracellular DNA analysis [0049] 18. 7-AAD:
7-aminoactinomycin [0050] 19. FCS: Fetal bovine serum [0051] 20.
CD34+Sca-1+c-kit+lin- cells: Primitive murine hematopoietic stem
cells that have full and long-term hematopoietic reconstitution
potential in vivo. [0052] 21. Flt3 (Rosnet et al. Oncogene, 6,
1641-1650, 1991) and flk-2 (Matthews et al., Cell, 65, 1143-1152,
1991) are variant forms of a TKR that is related to the c-fms and
c-kit receptors. The flk-2 gene product is expressed on
hematopoietic and progenitor cells, while the flt3 gene product has
a more general tissue distribution. The flt3 and flk-2 receptor
proteins are similar in amino acid sequence and vary at two amino
acid residues in the extracellular domain and diverge in a 31 amino
acid segment located near the C-termini (Lyman et al., Oncogene, 8,
815-822, 1993). [0053] 22. Flt3-ligand ("flt3-L") has been found to
regulate the growth and differentiation of progenitor and stem
cells and is likely to possess clinical utility in treating
hematopoietic disorders, in particular, aplastic anemia and
myelodysplastic syndromes. Additionally, flt3-L will be useful in
allogeneic, syngeneic or autologous bone marrow transplants in
patients undergoing cytoreductive therapies, as well as cell
expansion. Flt3-L will also be useful in gene therapy and
progenitor and stem cell mobilization systems.
Examples
Isolation and Culture of Primary Human Brain Endothelial Cells
(HUBEC)
[0054] Short segments (<10 cm) of blood vessels contained within
the central nervous system (segments of the anterior cerebral
artery and vertebro-basilar artery branching from the Circle of
Willis) and segments of vessels from outside the CNS (internal
iliac artery and renal artery) were obtained from autopsy specimens
less than 12 hours post-mortem after informed consent was obtained.
Blood vessel segments were placed in 4.degree. C. complete
endothelial cell culture medium consisting of M199 (Gibco BRL,
Grand Island, N.Y.) supplemented with 10% heat-inactivated FBS
(Hyclone, Logan, Utah), 100 mcg/mL L-glutamine, 50 mcg/mL heparin,
30 mcg/mL endothelial cell growth factor supplement (Sigma, St.
Louis, Mo.) and 100 mcg/mL penicillin/streptomycin solution.
[0055] Within 6 hours of primary dissection from the brain, blood
vessels were gently washed twice with PBS (Ca2+, Mg2+ free) and
transferred to gelatin-coated (need size) tissue culture dishes
containing 2 mL of complete endothelial cell growth media. Using a
sterile #10 scalpel blade, 1 mm cross sectional cuts were made
along the length of the vessels. Larger vessels were first cut
longitudinally with three incisions, to open and flatten the
vessel, and then inverted to orient the vessel lumen towards the
surface of the tissue culture dish. Immediately following the
dissection an additional 2 mL of complete endothelial cell media
was added to each dish. Cultures were placed in a humidified
37.degree. C., 5% CO.sub.2 atmosphere.
[0056] Distinct macroscopic cobblestone HUBEC colonies were evident
between days 7-14 of culture. Following the establishment of
confluent monolayers (-30 days), spent culture medium was collected
and endothelial cell monolayers were washed vigorously with PBS
(Ca++, Mg++Free), trypsinized (0.25 mg trypsin/mL, 5 mmol/L EDTA,
37.degree. C., 10 minutes; GIBCO) and subcultured at a ratio of 1:5
into gelatin-coated 75 cm2 flasks (Costar, Cambridge, Mass.)
containing 20 mL of complete endothelial cell culture medium. HUBEC
monolayers were fed weekly with complete medium and several
passages of the primary cells were established and banked.
Characteristics of Human Brain Endothelial Cells
[0057] HUBEC from passages 1-10 appeared morphologically identical
with no observable differences in the rate of growth noted.
Cultures developed the typical uniform endothelial cell monolayer
cobblestone morphology when 80-100% confluent (FIG. 1A). Cells at
passages 5-10 were harvested using 5 mM EDTA and stained with a
monoclonal antibody against human Von Willebrand Factor, and then
analyzed by flow cytometry. As shown in FIG. 1B, Von Willebrand
Factor is highly expressed on HUBEC. HUBEC do not express either
the CD34 or CD38 antigen at significant levels (<5%, data not
shown).
Expansion of Bone Marrow CD34+ Cells on HUBEC Monolayers
[0058] Human CD34.sup.+ cells were isolated from normal human
cadaveric bone marrow as previously described [21] with >96%
purity. The effects of HUBEC co-culture on CD34.sup.+ cell
proliferation and CFC generation were initially compared with
stroma-free liquid suspension cultures and co-cultures utilizing
human non-brain endothelial cells isolated from the same cadaveric
donors. All cultures were treated identically with a combination of
five stimulatory cytokines (GM-CSF+IL-3+IL-6+SCF+flt-3 ligand)
previously shown to support optimal CD34.sup.+ cell proliferation
[21]. After 7 days of co-culture, large macroscopic colonies
(>2000 cells) developed on HUBEC in which the majority of the
cells could be dispersed and collected by gently washing of the
HUBEC monolayers with culture medium. Remaining cells (<10%)
appeared to be tightly adherent and embedded within the HUBEC
monolayer resembling "cobblestone-like hematopoietic foci" (FIGS.
2A & 2B). In the absence of exogenously supplied growth factors
very little cell growth was observed. At day 7 of HUBEC coculture,
the mean number of total nonadherent and CD34.sup.+ cells increased
13.4- and 6.4-fold, respectively, with a 453-fold increase in the
number of CD34.sup.+CD38.sup.- cells (Table 1). Forty-eight percent
of the harvested nonadherent cells following 7 days of HUBEC
coculture expressed the CD34 antigen. The CD34.sup.+CD38.sup.-
subpopulation, defined as CD34.sup.+ cells that expressed CD38PE
fluorescence at least one half less than the PE-isotype control,
increased from a mean of 0.3% of the population at day 0 to 10.5%
of the total nonadherent cell population at day 7 and constituted
21% of the day 7 expanded CD34.sup.+ cell pool (Table 1). FIGS. 3A
and 3B show a representative phenotype of bone marrow CD34.sup.+
cells at day 0 (3A) and after 7 days of HUBEC co-culture (3B). As
shown in FIG. 4, CD34.sup.+CD38.sup.- cells isolated by cell
sorting from day 7 HUBEC co-cultures are primarily agranular blasts
with a high nuclear to cytoplasmic ratio, a fine chromatin pattern,
and prominent nucleoli.
[0059] We also compared the capacity of the HUBEC coculture system
to expand CD34.sup.+CD38.sup.- cells and multilineage CFC with
stroma-free liquid suspension cultures and with non-brain
endothelial cell cocultures using the identical combination of
exogenous cytokines over 7-14 days of culture. Maximal nonadherent
(233-fold) and total CD34.sup.+ cell expansion (21-fold) was
detected following 14 days of culture using the HUBEC coculture
system (Table 2), with a 1690-fold increase in the absolute number
of CD34.sup.+CD38.sup.- cells. Additionally, CFU-GM, CFU-Mix and
BFU-E CFC progenitors increased 558-, 129-, and 180-fold
respectively (Table 3). In comparison, overall cell and CFC yields
were significantly lower in stroma-free liquid suspension and in
non-brain endothelial cell co-cultures (Tables 2 and 3). Total
CD34.sup.+ cell numbers were maintained or moderately increased
(.ltoreq.7-fold) over 14 days under these culture conditions with
little or no amplification of the CD34.sup.+CD38.sup.- cell
population detected following 7 days of ex vivo culture.
Representative day 7 phenotypes of hematopoietic cells expanded in
liquid suspension cultures and non-brain endothelial cell
cocultures are shown in FIGS. 3C and 3D.
TABLE-US-00001 TABLE 1 Ex-vivo Expansion of Human Bone Marrow
CD34.sup.+ and CD34.sup.+CD38.sup.- Cells in Cytokine-Treated HUBEC
Cultures No. of Cells Procured .times. 10.sup.5 Culture Conditions
Cell yield .times. 10.sup.5 CD34.sup.+ CD34.sup.+CD38.sup.+
CD34.sup.+CD38.sup.- Input CD34.sup.+ Cells 5.0 5.0 4.85 0.015
(100) (99.7) (0.3) HUBEC Co-culture (day 7) 67 .+-. 17.1 32 .+-.
7.0 25.3 .+-. 7.4 6.8 .+-. 3.9 (100) (79) (21) CD34.sup.+ BM cells
(5 .times. 10.sup.5) were plated per culture treatment. Nonadherent
cells were procured on day 7 of culture. Cells of each culture were
stained for phenotypic analysis with FITC-conjugated CD34 (HPCA-2)
plus PE-conjugated CD38). Stained cells were analyzed using
two-color flow cytometry. The number of each immunophenotype was
corrected to reflect the total number of cells procured/culture.
Each point represents the mean number of positive cells from five
different experiments. Numbers in parentheses indicate the relative
frequency of a given phenotype calculated as a percentage of total
CD34.sup.+ cells.
TABLE-US-00002 TABLE 2 In Vitro Expansion of CD34.sup.+ Cell
Subsets in HUBEC Coculture versus Stroma-free and Non-brain
Endothelial Cell Cocultures No. of Cells Procured .times. 10.sup.5
Culture Conditions Cell yield .times. 10.sup.5 CD34.sup.+
CD34.sup.+CD38.sup.+ CD34.sup.+CD38.sup.- Input 5.0 5.0 4.99 + 0.01
0.01 + 0.01 HUBEC Co-culture Day 7 72.3 .+-. 2.6 33.0 .+-. 1.0 28.9
.+-. 0.9 4.4 .+-. 0.2 Day 14 1163.0 .+-. 43.6 104.3 .+-. 57.0 87.3
.+-. 5.0 16.9 .+-. 1.0 Stroma-free Day 7 51.0 .+-. 1.3 15.1 .+-.
0.7 15.1 .+-. 0.6 0.02 .+-. 0.02 Day 14 396.0 .+-. 80.0 35.0 .+-.
2.8 35.0 .+-. 2.8 0 Non-CNS EC Co-culture Day 7 52.0 .+-. 2.8 18.1
.+-. 12.6 18.1 .+-. 12.6 0 Day 14 nd nd nd nd CD34.sup.+ BM cells
(5 .times. 10.sup.5) were plated per culture treatment.
Non-adherent cells were procured on day 7 and 14 of culture. Cells
of each culture were stained for phenotypic analysis with
FITC-conjugated CD34 (HPCA-2) plus PE-conjugated CD38). Stained
cells were analyzed using two-color flow cytometry. The number of
each immunophenotype was corrected to reflect the total number of
cells procured/culture. Each point represents the mean number of
positive cells from two different experiments. nd: no data
TABLE-US-00003 TABLE 3 Effects of HUBEC Co-culture on Hematopoietic
Progenitor Cell Production in Comparison to Stroma-free and Human
Non-brain Endothelial Cell Co-cultures. Number of CFC .times.
10.sup.4 -- Culture Conditions CFU-GM CFU-Mix BFU-E Total CFC Input
3.7 + 0.9 0.5 + 0.2 0.6 + 0.3 4.7 + 1.4 HUBEC Co-culture Day 7 56.0
.+-. 2.2 2.6 .+-. 0.3 4.8 .+-. 0.6 63.4 .+-. 2.5 Day 14 2065.0 .+-.
49.0 64.5 .+-. 2.5 108.0 .+-. 9.5 2240.0 .+-. 70.7 Stroma-free Day
7 15.8 .+-. 4.5 0.5 .+-. 0.9 0.7 .+-. 0.7 17.1 .+-. 4.6 Day 14
200.0 .+-. 42.4 3.6 .+-. 0.2 19.8 .+-. 2.6 228.0 .+-. 41.0 Non-CNS
EC Co-culture Day 7 22.2 .+-. 4.9 0.6 .+-. 0.3 4.8 .+-. 0.3 27.7
.+-. 6.2 Day 14 nd nd nd nd 5 .times. 10.sup.5 CD34.sup.+ bone
marrow cells were plated per culture treatment. Nonadherent cells
were harvested on day 7 of culture. Nonadherent cells (5-500
.times. 10.sup.2) were cultured in 35-mm tissue culture dishes
containing IMDM medium, 1% methylcellulose, 30% FCS, optimal
concentrations of EPO, GM-CSF, IL-3 and SCF. The number of myeloid
and erythroid colonies were counted after 14 days of culture, and
based on the total number of viable cells per culture the number of
colonies was corrected to reflect the total number of CFC per
culture condition. Values represent the number of colonies of
triplicate cultures from two different experiments. nd: no
data.
Cell Cycle Analysis
[0060] In another series of experiments, we studied the role of
HUBEC co-culture on the cell cycle status of ex-vivo expanded
CD34.sup.+ cells. Analysis of CD34.sup.+CD38.sup.- cells at day 0
demonstrated that 92.9% of the cells were in G.sub.0, 5.9% were in
G.sub.1, and 1.2% were in G.sub.2/S/M phase (FIG. 5A). After 7 days
of HUBEC co-culture, 55.2% of the CD34.sup.+ CD38.sup.- cells had
entered G.sub.1, 38.7% were in G.sub.2/S/M phase, and only 5.8%
remained in G.sub.0 (FIG. 5B). Similar analysis of the
CD34.sup.+CD38.sup.+ subset indicated that 29.8%, 52.5%, and 17.2%
of the CD38.sup.+ cells were in G.sub.0, G.sub.1, and G.sub.2/S/M
phase at day 7, respectively. Analysis of CD34.sup.+ cells from
stroma-free and non-CNS endothelial cell cultures was not performed
due to the relatively low/undetectable frequency of
CD34.sup.+CD38.sup.- cells following 7 days of culture.
Effect of Ex Vivo HUBEC Co-Culture on the Clonogenic Capacity of
CD34.sup.+CD38.sup.- Cells in Methylcellulose CFC Cultures
[0061] To determine whether 7 days of HUBEC co-culture could
enhance the in vitro clonogenic capacity of CD34.sup.+CD38.sup.-
cells, we FACS sorted and collected CD34.sup.+,
CD34.sup.+CD38.sup.-, and CD34.sup.+CD38.sup.+ cell populations
prior to and following 7 days of HUBEC co-culture.
CD34.sup.+CD38.sup.- and CD34.sup.+CD38.sup.+ cells could be easily
collected in all samples analyzed. Sort windows were established to
give a clear separation of CD34.sup.+CD38.sup.- and
CD34.sup.+CD38.sup.bright cells, and therefore most of the
CD34.sup.+CD38.sup.dim cells were excluded from the analysis. Five
hundred cells from each cell population were seeded into 1%
methylcellulose containing Iscove's modified Dulbecco's medium
(IMDM), supplemented with optimal concentrations of EPO, GM-CSF,
IL-3, IL-6, and SCF and scored for total CFC formation after 14
days of incubation. As shown in Table 4 and consistent with
previous studies [29,30], very few, if any, steady state
CD34.sup.+CD38.sup.- cells (0.035%) were able to form colonies in
standard methylcellulose based clonogenic media. Colonies derived
from CD34.sup.+CD38.sup.- cells were smaller on average than
colonies derived from steady state CD34.sup.+CD38.sup.+ cells
(cloning efficiency 11.9%) cultured under identical culture
conditions. As expected, the CD34.sup.+CD38.sup.+ subset
demonstrated a cloning efficiency which approximated the cloning
efficiency of the entire steady state CD34.sup.+ population
(consisting of .about.98% CD34.sup.+CD38.sup.+ cells), thereby
confirming that the large majority of colonies generated from
steady state CD34.sup.+ cells arise from the CD34.sup.+CD38.sup.+
subset with little or no contribution from the CD34.sup.+CD38.sup.-
subset.
[0062] In contrast to the results obtained culturing steady state
CD34.sup.+CD38.sup.- progenitor cells, when activated/expanded
CD34.sup.+CD38.sup.- cells were stringently re-selected from HUBEC
monolayers after 7 day of coculture, a 685-fold expansion (from
0.035% cloning efficiency at day 0 to 24.0% cloning efficiency at
day 7) of CFC was detected. The number of assayable CFC was greater
in cultures initiated with CD34.sup.+CD38.sup.- cells (24.0%
cloning efficiency) than in cultures initiated with
CD34.sup.+CD38.sup.bright cells (16.8%), but lower when compared to
unsorted CD34.sup.+ cells (40.1%). This is most likely due to the
fact that CD34.sup.+CD38.sup.dim cells which have a high clonogenic
potential comprise a significant portion of the day 7 CD34.sup.+
cell pool and these were excluded from our analysis in the setting
of stringent sort windows. In addition to increased colony numbers,
an increase in colony size was also observed for cultures initiated
with expanded and sorted CD34.sup.+CD38.sup.- in comparison to
CD34.sup.+CD38.sup.+ cells. Evaluation of the sorted CD34.sup.-
cells from HUBEC co-cultures showed that this population was
practically devoid of CFC (0.3%). These plating efficiencies
indicate that the majority of CFC generated following 7 days of
HUBEC co-culture arises from the CD34.sup.+CD38.sup.- and
CD34.sup.+CD38.sup.dim populations with significantly less
contribution from the CD34.sup.+CD38.sup.bright subset.
TABLE-US-00004 TABLE 4 Frequency of CFC in Sorted CD34.sup.+ Cell
Populations CFC Frequency (%) Cell Population Experiment 1
Experiment 2 Mean Input CD34.sup.+ Cells (d-0) Unsorted CD34.sup.+
13. 4 .+-. 4.1 12.8 .+-. 3.7 13.1 CD34.sup.+CD38.sup.- 0.07 .+-.
0.01 0 0.035 CD34.sup.+CD38.sup.+ 11.4 .+-. 3.5 12.4 .+-. 4.3 11.9
HUBEC Co-culture (d-7) 19.9 .+-. 1.1 19.9 .+-. 1.9 19.9 Unsorted
nonadherent cells CD34.sup.+ 42.2 .+-. 2.6 37.9 .+-. 1.2 40.1
CD34.sup.+CD38.sup.- 28.9 .+-. 5.3 19.1 .+-. 2.8 24.0
CD34.sup.+CD38.sup.+ 17.1 .+-. 4.6 16.4 .+-. 1.7 16.8 CD34.sup.-
0.2 .+-. 0.2 0.4 .+-. 0.1 0.3 At day 0 and after 7 days of HUBEC
co-culture CD34.sup.+, CD34.sup.+CD38.sup.+, CD34.sup.+CD38.sup.-,
and CD34.sup.- cell populations were collected by FACS sorting
cells labeled with FITC-conjugated anti-human CD34 mAb and
PE-conjugated anti-human CD38 mAb. Cells were cultured at 500
cells/dish in 1% methylcellulose containing Iscove's modified
Dulbecco's medium (IMDM), supplemented with optimal concentrations
of EPO, GM-CSF, IL-3, and SCF. The cultures were assessed at day 14
for colony-forming cells (CFC). Based on the total number of viable
cells per culture the number of colonies was corrected to reflect
the total number of CFC per culture condition. Values represent the
number of colonies of triplicate cultures from two different
experiments.
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[0104] Obviously, many variations and combinations of the invention
can be seen from the above specific examples. The above examples
are intended to disclose the best mode currently known to the
inventors and is not intended to limit the invention.
* * * * *