Method for delivering a biological compound using neural progenitor cells
derived from whole bone marrow
United States Patent: 7,504,100
Issued: March 17, 2009
Inventors: Yu; John S. (Los
Angeles, CA), Kabos; Peter (Los Angeles, CA), Ehtesham; Moneeb (Nashville,
Medical Center (Los Angeles, CA)
Appl. No.: 11/364,394
Filed: February 28, 2006
Covidien Pharmaceuticals Outsourcing
A method is described for generating a
clinically significant volume of neural progenitor cells from whole bone
marrow. A mass of bone marrow cells may be grown in a culture supplemented
with fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF).
Further methods of the present invention are directed to utilizing the
neural progenitor cells cultured in this fashion in the treatment of
various neuropathological conditions, and in targeting delivery of cells
transfected with a particular gene to diseased or damaged tissue.
Description of the
FIELD OF THE INVENTION
Embodiments of the present invention are directed to a method for
generating a clinically substantial volume of neural progenitor cells from
mammalian whole bone marrow. Further embodiments of the present invention
are directed to the treatment of neurological disorders using neural
progenitor cells cultured in this fashion.
BACKGROUND OF THE INVENTION
Nearly every cell in an animal's body, from neural to blood to bone, owes
its existence to a stem cell. A stem cell is commonly defined as a cell
that (i) is capable of renewing itself; and (ii) can give rise to more
than one type of cell (that is, a differentiated cell) through asymmetric
cell division. F. M. Watt and B. L. M. Hogan, "Out of Eden: Stem Cells and
Their Niches," Science, 284, 1427-1430 (2000). Stem cells give rise to a
type of stem cell called progenitor cells; progenitor cells, in turn,
proliferate into the differentiated cells that populate the body.
The prior art describes the development, from stem cell to differentiated
cells, of various tissues throughout the body. U.S. Pat. No. 5,811,301,
for example, the disclosure of which is hereby incorporated by reference,
describes the process of hematopoiesis, the development of the various
cells that comprise blood. The process begins with what may be a
pluripotent stem cell, a cell that can give rise to every cell of an
organism (there is only one cell that exhibits greater developmental
plasticity than a pluripotent stem cell; this is a fertilized ovum, a
single, totipotent stem cell that can give rise to an entire organism when
implanted into the uterus). The pluripotent stem cell gives rise to a
myeloid stem cell. Certain maturation-promoting polypeptides cause the
myeloid stem cell to differentiate into precursor cells, which in turn
differentiate into various progenitor cells. It is the progenitor cells
that proliferate into the various lymphocytes, neutrophils, macrophages,
and other cells that comprise blood tissue of the body.
This description of hematopoiesis is vastly incomplete, of course: biology
has yet to determine a complete lineage for all the cells of the blood
(e.g., it is has yet to identify all the precursor cells between the
myeloid stem cell and the progenitor cells to which it gives rise), and it
has yet to determine precisely how or why the myeloid cell differentiates
into progenitor cells. Even so, hematopoiesis is particularly well
studied; even less is known of the development of other organ systems.
With respect to the brain and its development, for example, U.S. Pat. No.
6,040,180, the disclosure of which is hereby incorporated by reference,
describes the "current lack of understanding of histogenesis during brain
development." U.S. Pat. No. 5,849,553, the disclosure of which is hereby
also incorporated by reference, describes the "uncertainty in the art
concerning the development potential of neural crest cells."
The identification and isolation of stem cells has daunted researchers for
decades. To date, no one has identified an individual neural stem cell or
hematopoietic stem cell. F. H. Gage, "Mammalian Neural Stem Cells,"
Science, 287, 1433-1488 (2000). There are two principal difficulties.
First, stem cells are rare. In bone marrow, for example, where
hematopoiesis occurs, there is only one stem cell for every several
billion bone marrow cells. G. Vogel, "Can Old Cells Learn New Tricks?"
Science, 287, 1418-1419 (2000). Second, and more importantly, researchers
have been unable to identify molecular markers which are unique to stem
cells; to the typical immunoassay, most stem cells look like any other
cell. Id. Compounding this problem is that primitive stem cells may be in
a quiescent state. As a result, they may express few molecular markers. F.
H. Gage, supra.
A method to effectively isolate stem cells and culture them in clinically
significant quantities would be of immense importance. Researchers are
already transplanting immature neurons, presumed to contain neural stem
cells, from human fetuses to adult patients with neurodegenerative
disease. The procedure has reduced symptoms by up to 50% in patients with
Parkinson's disease in one study. M. Barinaga, "Fetal Neuron Grafts Pave
the Way for Stem Cell Therapies," Science, 287, 1421-1422 (2000). Many of
the shortcomings of this procedure, including the ethical and practical
difficulties of using material derived from fetuses and the inherent
complications of harvesting material from adult brain tissue, could be
addressed by using cultures of isolated stem cells, or stem cells obtained
from adult individuals. D. W. Pincus et al., Ann. Neurol. 43:576-585
(1998); C. B. Johansson et al., Exp. Cell. Res. 253:733-736 (1999); and S.
F. Pagano et al., Stem Cells 18:295-300 (2000). However, the efficient and
large-scale generation of neural progenitor cells for use in the treatment
of neurological disorders has been a challenge.
Recent evidence has suggested that progenitor cells outside the central
nervous system and bone marrow cells in paricular may have the ability to
generate either neurons or glia in vivo. J. G. Toma et al., Nat. Cell
Biol. 3:778-783 (2001); E. Mezey et al., Science 290:1779-1782 (2000); T.
R. Brazleton et al., Science 290:1775-1779 (2000); and M. A. Eglitis et
al., Proc Natl. Acad. Sci. 94:4080-4085 (1997). Bone marrow stromal cells
have also been shown to be capable of differentiating into neurons and
glia in vitro after a complicated and time-consuming culture process
spanning several weeks. The generation of neural progenitor cells from
whole bone marrow has, however, not been reported.
SUMMARY OF THE INVENTION
The invention described herein provides an efficient method of generating
a clinically significant quantity of neural progenitor cells. These neural
progenitor cells may be generated from bone marrow or other appropriate
sources, and may be used to treat a variety of conditions, particularly
neuropathological conditions. Owing to the neural progenitor cells'
ability to track diseased or damaged neural tissue and to further replace
the lost function of such tissue, the cells of the present invention are
particularly useful in the treatment of conditions wherein neural tissue
itself is damaged.
Still further embodiments of the present invention describe the use of the
neural progenitor cells to target the delivery of various compounds to
damaged or diseased neural tissue. Neural progenitor cells may be caused
to carry a gene that induces the cells themselves to secrete such
compounds, or to otherwise effect the local production of such compounds
by, for example, initiating or promoting a particular biochemical pathway.
Since the neural progenitor cells that carry these genes may track
diseased or damaged neural tissue, delivery of the particular compound may
be correspondingly targeted to such tissue. A dual treatment effect is
accomplished when the neural progenitor cells both replace lost or damaged
neural tissue function while simultaneously effecting the targeted
delivery of a therapeutic compound.
DETAILED DESCRIPTION OF THE INVENTION
Methods of the present invention are based on adult bone marrow as a
viable alternative source of neural progenitor cells that may be used in
therapeutic strategies for a variety of neuropathological conditions.
Any population of cells where neural progenitor cells are suspected of
being found may be used in accordance with the method of the present
invention. Such populations of cells may include, by way of example,
mammalian bone marrow, brain tissue, or any suitable fetal tissue.
Preferably, cells are obtained from the bone marrow of a non-fetal mammal,
and most preferably from a human. U.S. Pat. Nos. 6,204,053 B1 and
5,824,489, the disclosures of which are hereby incorporated by reference,
identify additional sources of cells that contain or are thought to
contain stem cells; any of these cells may be used in accordance with the
methods of the present invention.
In one embodiment of the present invention, a mass of cells may be
harvested or otherwise obtained from an appropriate source, such as, by
way of example, adult human bone marrow. The mass of cells may thereafter
be grown in a culture, and may be further subcultured where desirable, to
generate further masses of cells. Any appropriate culture medium may be
used in accordance with the methods of the present invention, such as, by
way of example, serum-free Dulbecco's modified Eagle medium (DMEM)/F-12
The medium of the present invention may include various medium
supplements, growth factors, antibiotics, and additional compounds.
Supplements may illustratively include B27 supplement and/or N2 supplement
(both available from Invitrogen Corporation); growth factors may
illustratively include fibroblast growth factor-2 (FGF-2), epidermal
growth factor (EGF), and/or leukemia inhibitory factor (LIF); and
antibiotics may illustratively include penicillin and/or streptomycin. In
preferred embodiments of the present invention, growth factors are
included in an amount of from about 15 ng/ml to about 25 ng/ml. Additional
compounds suitable for use in the present invention may include, but are
in no way limited to, interleukin-3 (IL-3), stem cell factor-1 (SCF-1),
sonic hedgehog (Shh), and fms-like tyrosine kinase-3 (Flt3) ligand. While
not wishing to be bound by any theory, it is believed that these
particular compounds may enhance the production of spheres in accordance
with the methods of the present invention. Additional or substituted
supplements, growth factors, antibiotics, and additional compounds
suitable for use with the methods of the present invention may be readily
recognized by one of skill in the art, and these are contemplated as being
within the scope of the present invention. In a most preferred embodiment
of the present invention, a culture medium is DMEM/F-12 medium
supplemented with B27, and additionally includes 10 ng/ml of both FGF-2
and EGF, as well as penicillin and streptomycin.
After a sufficient time period (generally from about three to about six
days), clusters of neural progenitor cells (e.g., spheres) may form in a
culture medium in which stem cells obtained as described above are
included. Individual clusters of neural progenitor cells may be removed
from the medium and sub-cultured separate from one another. Such
separation may be repeated any desirable number of times to generate a
clinically significant volume of neural progenitor cells. These neural
progenitor cells may be capable of differentiating into a variety of
neural cells, such as, astrocytes, neurons, and oligodendroglia.
As used herein, a "clinically significant volume" is an amount of cells
sufficient to utilize in a therapeutic treatment of a disease condition,
including a neuropathological condition. Furthermore, as used herein,
"treatment" includes, but is not limited to, ameliorating a disease,
lessening the severity of its complications, preventing it from
manifesting, preventing it from recurring, merely preventing it from
worsening, mitigating an undesirable biologic response (e.g.,
inflammation) included therein, or a therapeutic effort to effect any of
the aforementioned, even if such therapeutic effort is ultimately
The neural progenitor cells of the present invention possess a host of
potential clinical and therapeutic applications, as well as applications
in medical research. Two possible therapeutic mechanisms include: (1%)
using the cells as a delivery vehicle for gene products by taking
advantage of their ability to migrate after transplantation, and (2) using
the cells to replace damaged or absent neural tissue, thereby restoring or
enhancing tissue function.
As discussed in the ensuing Examples, and with reference to the first
therapeutic mechanism indicated above, the neural progenitor cells of the
present invention are capable of "tracking" diseased or damaged tissue in
vivo. The cells may therefore be used to aid in the targeted delivery of
various compounds useful in the treatment of diseased or damaged tissue.
Delivery of such compounds may be accomplished by transfecting the cells
with a gene that induces the cell to, for example, constitutively secrete
that compound itself, or promote a biochemical pathway that effects a
local production of that compound.
Thus, in one embodiment of the present invention, neural progenitor cells
may be transfected with or otherwise caused to carry a particular gene, as
per any conventional methodology. Such methodologies may include
introducing a particular gene into the neural progenitor cells as a
plasmid, or, more preferably, using somatic cell gene transfer to
transfect the cells utilizing viral vectors containing appropriate gene
sequences. Suitable viral vectors may include, but are in no way limited
to, expression vectors based on recombinant adenoviruses, adeno-associated
viruses, retroviruses or lentiviruses, although non-viral vectors may
alternatively be used. In a preferred embodiment of the present invention,
one employs adenovirus serotype 5 ("Ad5")-based vectors (available from
Quantum Biotechnology, Inc., Montreal, Quebec, Canada) to deliver and
express desirable gene sequences in the neural progenitor cells of the
present invention. Once caused to carry the desired gene, the neural
progenitor cells may be implanted in or otherwise administered to a
By employing this therapeutic mechanism, the neural progenitor cells of
the present invention may be used to treat a variety of pathological
conditions; potentially any condition where mammalian neural tissue is
diseased or damaged to the point that neural progenitor cells will track
the same. In the area of neuropathological disorders, this therapeutic
modality may be used in the treatment of numerous conditions, some
examples of which may include: brain tumors (e.g., by targeting the
delivery of cytokines or other agents that enhance the immune response, or
by targeting the delivery of compounds that are otherwise toxic to tumor
cells); brain ischemia (e.g., by targeting the delivery of neuroprotective
substances such as brain-derived neurotrophic factor (BDNF), nerve growth
factor (NGF), and neurotrophin-3, -4, and -5 (NT-3, NT-4, NT-5)); spinal
cord injury (e.g., again, by targeting the delivery of neuroprotective
substances, or by targeting the delivery of substances inducing neurite
growth such as basic fibroblast growth factor (bFGF), insulin-like growth
factor-1 (IGF-1), and glial-derived neurotrophic factor (GDNF)); and
neurodegenerative disorders, such as Alzheimer's or Parkinson's Disease
(e.g., again, by targeting the delivery of neuroprotective substances or
growth factors, or by targeting the delivery of other neuroprotective
factors such as amyloid precursor proteins or protease nexin-1).
As discussed in the ensuing Examples, and with reference to the second
therapeutic mechanism indicated above, the neural progenitor cells of the
present invention are also able to replace neurons and glia in vivo. The
cells may therefore be used to replace diseased or damaged neural tissue,
and, owing to the cells' additional capacity to track diseased or damaged
tissue in vivo, once administered, the cells may configure themselves to
an appropriate physiological site to effect this therapeutic mechanism.
Given the ability of the neural progenitor cells of the present invention
to replace lost or damaged neural tissue function, these cells may be
useful in the treatment of numerous neuropathological conditions, many of
which are similar to those enumerated above. By way of example, even in a
state where the cells have not been transfected or otherwise caused to
carry a particular gene, the cells may be used in the treatment of brain
tumors, brain ischemia, spinal cord injury, and various neurodegenerative
Neural progenitor cells that are, in fact, transfected or otherwise caused
to carry a desirable gene may also provide the additional neural cell
function replacement capacity discussed in this mechanism; thereby
imparting a dual treatment effect to the recipient. The dual treatment
effect may include the replacement of lost or damaged cell function (e.g.,
as per the second therapeutic mechanism) in conjunction with the targeted
delivery of a beneficial compound to that same region (e.g., as per the
first therapeutic mechanism). Therefore, in the illustrative instance of
brain tumor treatment, the neural progenitor cells may be transfected with
a gene that induces the secretion of cytokines (e.g., tumor necrosis
factor (TNF) or interleukin-1 (IL-1)), and implanted or otherwise
administered to the brain of a recipient. Once administered, the cells may
track the tissue damaged by the tumor, replacing at least a portion of the
lost brain function, while simultaneously secreting cytokines that may
induce an immune response against the tumor cells. This dual treatment
effect is further described in the ensuing Examples.
Neural progenitor cells developed through culture as described above may
be implanted in or otherwise administered to a mammal to effect the
therapeutic mechanisms previously discussed. Once implanted or otherwise
administered, these cells may relocate to an area of diseased tissue, such
as, but not limited to, brain tumors, tissue damaged by stroke or other
neurodegenerative disease, and the like. Moreover, the neural progenitor
cells may multiply in vivo, and may further follow diseased tissue as it
spreads (e.g., as a tumor spreads). Implantation may be performed by any
suitable method as will be readily ascertained without undue
experimentation by one of ordinary skill in the art, such as injection,
inoculation, infusion, direct surgical delivery, or any combination
Claim 1 of 22 Claims
1. A method to deliver a biological
compound, the method comprising: culturing whole bone marrow from a mammal
in a medium comprising fibroblast growth factor-2 (FGF-2) and epidermal
growth factor (EGF) to produce a neural progenitor cell; causing the
neural progenitor cell to carry a gene that effects local production of
the biological compound; and administering the neural progenitor cell to a
mammal to deliver the biological compound to a tumor or diseased neural
tissue, wherein the biological compound is a gene product.
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