U.S. patent application number 12/062230 was filed with the patent office on 2008-08-07 for cellular therapy to heal vascular tissue.
This patent application is currently assigned to Medtronic Vascular, Inc.. Invention is credited to Jack Chu, Brian Fernandes, Prema Ganesan.
Application Number | 20080187524 12/062230 |
Document ID | / |
Family ID | 33299056 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080187524 |
Kind Code |
A1 |
Fernandes; Brian ; et
al. |
August 7, 2008 |
Cellular Therapy to Heal Vascular Tissue
Abstract
The present invention encompasses methods and apparatus for
minimizing the risks inherent in endovascular grafting for blood
vessel therapy and repair. The invention involves delivering adult
stem cells, embryonic stem cells, progenitor cells, fibroblasts, or
smooth muscle cells to the diseased blood vessel.
Inventors: |
Fernandes; Brian;
(Roseville, MN) ; Chu; Jack; (Santa Rosa, CA)
; Ganesan; Prema; (San Francisco, CA) |
Correspondence
Address: |
MEDTRONIC VASCULAR, INC.;IP LEGAL DEPARTMENT
3576 UNOCAL PLACE
SANTA ROSA
CA
95403
US
|
Assignee: |
Medtronic Vascular, Inc.
Santa Rosa
CA
|
Family ID: |
33299056 |
Appl. No.: |
12/062230 |
Filed: |
April 3, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10423193 |
Apr 25, 2003 |
7387645 |
|
|
12062230 |
|
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Current U.S.
Class: |
424/93.21 ;
424/93.7 |
Current CPC
Class: |
A61F 2002/065 20130101;
A61P 7/00 20180101; A61F 2/07 20130101; A61F 2002/077 20130101 |
Class at
Publication: |
424/93.21 ;
424/93.7 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 7/00 20060101 A61P007/00 |
Claims
1-30. (canceled)
31. A method of treating an aneurysm in an individual at the
aneurysmal site using self-derived cells, comprising: harvesting
adult stem cell-containing tissue from the individual wherein said
tissue is selected from the group consisting of adipose tissue,
bone marrow and peripheral blood; isolating adult stem cells from
the tissue; and delivering the isolated adult stem cells directly
to the wall of a blood vessel at said aneurysmal site by liquid
delivery means wherein said blood vessel wall is repaired and said
aneurysm is treated in said individual.
32. The method of claim 31, wherein the harvesting step is
accomplished by liposuction, drawing blood, or harvesting bone
marrow.
33. The method of claim 31, wherein the adult stem cells are
isolated by growth in selective medium, by density centrifugation,
fluorescence activated cell sorting or by magnetic activated cell
sorting.
34. The method of claim 31, wherein the adult stem cells are
isolated by assaying for adult stem cell-specific a actin,
calponin, smooth muscle myosin heavy chain, .beta.-tubulin or
vimentin expression.
35. The method of claim 31, further comprising the step of
modifying the adult stem cells after the isolating step.
36. The method of claim 35, wherein the modifying step is
accomplished by genetic engineering.
37. The method of claim 36, wherein the adult stem cells are
genetically engineered to produce at least one cellular factor
selected from the group of collagen.
Description
BACKGROUND OF THE INVENTION
[0001] Aortic aneurysms and the degeneration of the vasculature in
general represent a significant medical problem for the general
population. Aneurysms within the aorta presently affect between two
and seven percent of the general population and the rate of
incidence appears to be increasing. This form of vascular disease
is characterized by degeneration in the arterial wall in which the
wall weakens and balloons outward. Until the affected artery is
grafted through open repair or treated with a stent graft
endovascularly, a patient with an aortic aneurysm must live with
the threat of aortic aneurysm rupture and death.
[0002] One known clinical approach for patients with an aortic
aneurysm is a surgical repair procedure. This is an extensive
operation involving dissection of the aorta and reinforcement of
the aneurysm wall with a prosthetic graft.
[0003] Alternatively, there is a significantly less invasive
clinical approach to aneurysm repair known as endovascular
grafting. Endovascular grafting involves the placement of a
prosthetic arterial stent graft within the lumen of the artery. To
prevent rupture of the aneurysm, a stent graft of tubular
construction is introduced into the blood vessel, and is secured in
a location such that the stent graft spans the length of the
aneurysmal sac. The outer surface of the stent graft at its ends is
sealed to the interior wall of the blood vessel at a location where
the blood vessel wall has not suffered a loss of strength or
resiliency, such that blood flowing through the vessel is diverted
through the hollow interior of the stent graft away from the blood
vessel wall at the aneurysmal sac location. In this way, the risk
of rupture of the blood vessel wall at the aneurysmal location is
significantly reduced and blood can continue to flow through to the
downstream blood vessels without interruption. However, despite the
advantages of endovascular grafting over other surgical procedures,
there is, nonetheless, continued progression of the aneurysm
disease.
[0004] A salient feature of aneurysm formation is the gradual
degradation of extracellular components, such as collagen and
elastin, as well as the loss of resident cells, namely smooth
muscle cells and fibroblasts. The cells in a healthy vessel perform
many and varied functions, including providing reinforcement to the
vessel wall and, importantly, replenishing the extracellular
components. The diminished cellular presence observed in diseased
arteries directly and adversely impacts the vessel wall
ultrastructure.
[0005] The field of cell replacement research and tissue
engineering currently is one of the major focuses of medical
technology. An exciting area of tissue engineering is the emerging
technology of "self-cell" therapy, where cells of a given tissue
type are removed from a patient, isolated, perhaps mitotically
expanded and/or genetically engineered, and ultimately reintroduced
into the donor/patient with or without synthetic materials or other
carrier matrices. One goal of self-cell therapy is to help guide
and direct the rapid and specific repair or regeneration of
tissues. Such self-cell therapy is already a part of clinical
practice; for example, using autologous bone marrow transplants for
various hematologic conditions. The rapid advancement of this
technology is reflected in recent publications that disclose
progress toward bone and cartilage self-cell therapy. Moreover,
similar advances are being made with other tissues such as cardiac
muscle, liver, pancreas, tendon and ligament. One of the greatest
advantages of self-cell therapy over current technologies is that
the autologous nature of the tissue/cells greatly reduces, if not
eliminates, immunological rejection and the costs associated
therewith. [0006] Thus there is a desire in the art to slow,
reverse, or potentially cure the aneurysm disease state by using
minimally invasive procedures while reducing or eliminating
immunological rejection. The present invention satisfies this need
in the art.
SUMMARY OF THE INVENTION
[0007] The present technology addresses the problem of degeneration
of vascular tissue, particularly at an aneurysmal site. Embodiments
according to the present invention provide methods for supporting
and treating the vascular tissue with fully differentiated primary
cells, such as fibroblasts or smooth muscle cells, as well as stem
cells derived from adult sources. Such cells function to replace,
regenerate, reinforce and strengthen the disease site through the
secretion of extracellular matrix components in the tissue
wall.
[0008] Thus, in one embodiment, there is provided a method of
treating a blood vessel in an individual comprising: harvesting
tissue from the individual; isolating cells of one or more cell
type from the harvested tissue; and delivering the isolated cells
to the blood vessel by a delivery means. In one aspect, single cell
populations or combination cell populations (which include more
than one cell type) are used. In another aspect of the embodiment
of this invention, the isolated cells are expanded or
differentiated in vitro before delivery. In another aspect of the
embodiment of this invention, the cells are genetically engineered
in vitro before delivery. In another aspect of this embodiment of
the invention, the cells are delivered in conjunction with a
carrier and/or cellular scaffold and are left to expand, and, if
necessary, differentiate in vivo. In another aspect of the
embodiment, the cells are delivered together with agents, such as
growth factors, to promote or enhance cell proliferation and/or
secretion in vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] A more particular description of the invention, briefly
summarized above, may be had by reference to the embodiments of the
invention described in the present specification and illustrated in
the appended drawings. It is to be noted, however, that the
specification and appended drawings illustrate only certain
embodiments of this invention and are, therefore, not to be
considered to be limiting of its scope. The invention may admit to
equally effective embodiments as defined by the claims.
[0010] FIG. 1 is a schematic view of a human aortal aneurysm.
[0011] FIG. 2 is a flow chart of one embodiment of the methods of
the present invention.
[0012] FIG. 3 is a partial sectional view of a blood vessel with
vascular smooth muscle cells delivered thereto.
[0013] FIG. 4 is a partial sectional view of a descending aorta
with a bifurcated stent graft placed therein, and a delivery
catheter and stem cells delivered to an aneurismal sac.
DETAILED DESCRIPTION
[0014] Reference will now be made in detail to exemplary
embodiments of the invention. While the invention will be described
in conjunction with these embodiments, it is to be understood that
the described embodiments are not intended to limit the invention
solely and specifically to only these embodiments. On the contrary,
the invention is intended to cover alternatives, modifications, and
equivalents that may be included within the spirit and scope of the
invention as defined by the attached claims.
[0015] The present technology encompasses methods for treating the
aneurysm wall of blood vessels in an individual. Aspects of the
invention include a method for delivering adult stem cells,
including progenitor cells, derived from, for example, adipose
tissue, bone marrow, or peripheral blood of the individual, to a
blood vessel in need of therapy. Also included are methods for the
delivery of differentiated primary cells, such as vascular smooth
muscle cells or fibroblasts, derived from, for example, arterial or
venous blood vessel segments and dermal tissue (skin),
respectively. The stem cells, fibroblasts, or smooth muscle cells
to be delivered may come directly from the adipose tissue, bone
marrow, or other tissue samples, or the cells may be cultured,
expanded or manipulated before delivery. One cell type or a
combination of cell types may be delivered. In addition, the cells
may be delivered along with a natural or synthetic cellular
scaffolding material and/or carrier solution, and with or without
bioactive agents.
[0016] Referring initially to FIG. 1, there is shown generally an
aneurysmal blood vessel; in particular, there is an aneurysm of the
aorta 12, such that the aorta or blood vessel wall 04 is enlarged
at an aneurysmal site 14 and the diameter of the aorta 12 at the
aneurysmal site 14 is larger than the diameter of a healthy aorta
12. The aneurysmal site 14 forms an aneurysmal bulge or sac 18. If
left untreated, the aneurysmal sac 18 may continue to deteriorate,
weaken, increase in size, and eventually tear or burst.
[0017] As stated previously, vascular surgery and endovascular
grafts have proven successful in patients with aortic aneurysms;
however, neither procedure inhibits the progression of the disease
state. Before the aneurysm reaches a size to necessitate such
procedures, the present invention treats blood vessels
directly-particularly at the site of an aneurysm, possibly
preventing the need for subsequent surgical repair. Alternatively,
the present invention can be used in addition to surgery or
placement of a stent graft to bolster and provide enhanced healing
at the aneurysmal site. Such methods involve tissue engineering
using self-derived cells, or, alternatively, immuno-neutral
non-self-derived stem cells (for example, mesenchymal stem cells)
may be used.
[0018] Progression of the disease state is characterized by
continued degeneration of the aortic wall due to thinning of the
medial connective tissue architecture of the aorta, and a
concomitant loss of collagen in the adventitia associated with
dilatation of the vessel. There is evidence that connective
tissue-degrading enzymatic activity is triggered by inflammation in
the medial and adventitial layers of the aorta. Accordingly, one
aspect of one embodiment of the present invention involves limiting
the spread of inflammation by implantation of stem cells,
progenitor cells, or smooth muscle cells into the blood vessel wall
such that the newly created vascular smooth muscle exerts a
paracrine, protective effect against artery wall destruction by
inflammation. The smooth muscle cells and fibroblasts in this
normal in vivo setting thus are likely to synthesize aortic wall
collagen (type I & III) and elastin. Thus, among other
advantages, the invention provides the blood vessel with therapy
that protects and regenerates extracellular matrix components in
the medial and adventitial layers of blood vessels, preventing
continued dilatation and/or further degeneration of the aortic
tissue.
[0019] FIG. 2 is a flow chart of one embodiment of methods
according to the present invention. In FIG. 2, method 300 is
comprised of three main steps and two optional steps. In step 310,
tissue is harvested from, for example, adipose tissue, bone marrow,
blood or other tissues where stem cells and/or smooth muscle cells
may be found. For example, adipose tissue is readily accessible and
abundant in most individuals and can be harvested by liposuction.
Various liposuction techniques exist, including ultrasonic-assisted
liposuction ("UAL"), laser-assisted liposuction, and traditional
suction-assisted liposuction ("SAL"), where fat is removed with the
assistance of a vacuum created by either a mechanical source or a
syringe. Each of the foregoing liposuction techniques may be used
in conjunction with tumescent solution. Liposuction procedures that
use a tumescent solution generally involve pre-operative
infiltration of subcutaneous adipose tissue with large volumes of
dilute anesthetic solutions. The evolution of the tumescent
technique has revolutionized liposuction by making it available on
an outpatient basis. Specifically, it makes the use of general
anesthesia optional in most cases thereby avoiding the associated
risks and costs. (See, e.g., Rohrich, et al., Plastic and
Reconstructive Surgery, 99:514-19 (1997).)
[0020] Another advantage of using adipose tissue as a source of
stem cells is that, due to the abundance of stem cells in adipose
tissue, stem cell harvest, isolation, genetic manipulation and/or
growth-factor based differentiation may be accomplished
peri-operatively. Thus, depending on the number of cells required
for implantation, it may not be necessary for the patient to submit
to the liposuction procedure on one day and the stem cell
implantation on a subsequent day. The procedures can be performed
sequentially within minutes or tens of minutes of one another.
(See, e.g., Noishiki, et al., Artificial Organs, 25(3):228-35
(2001); and Zuk, et al., Tissue Engineering, 7(2):211-28).
[0021] Alternatively, bone marrow may be harvested for vascular
smooth muscle cell or fibroblast isolation. As a whole, bone marrow
is a complex tissue comprised of two distinct populations of stem
cells, namely hematopoietic stem cells and mesenchymal stem cells.
Hematopoietic stem cells give rise to components of the blood and
immune systems while mesenchymal stem cells give rise to varied
cells, including osteoblasts, chondrocytes, adipocytes,
fibroblasts, smooth muscle cells, and myoblasts. Cells, such as
fibroblasts, reticulocytes, adipocytes, and endothelial cells, form
a connective tissue network called "stroma". Cells from the stroma
regulate morphologically the differentiation of hematopoietic cells
through direct interaction via cell surface proteins and the
secretion of growth factors. Stroma cells also are involved in the
foundation and support of the bone structure. Studies using animal
models show that bone marrow contains "pre-stromal" cells which
have the capacity to differentiate into cartilage, bone, and other
connective tissue cells, and, in an inverse relationship with age,
they are capable of differentiating into an assortment of
connective tissues depending upon the influence of a number of
bioactive factors. While mesenchymal stem cells are extremely rare
in bone marrow, they also can be found in other tissues, such as
peripheral blood, umbilical cord blood and adipose tissue.
[0022] In certain embodiments according to the present invention,
an autologous bone marrow transplant is contemplated. In an
autologous transplant, the individual to receive therapy donates
his or her own stem cells for later reinfusion. The procedure for
harvesting bone marrow from the individual is performed while the
individual is under anesthesia. A needle is inserted into the
cavity of the rear hip bone, the iliac crest, where a large
quantity of bone marrow is located. The bone marrow is a thick, red
liquid and is extracted by a syringe. Several skin punctures on
each hip and multiple bone punctures may be required to harvest
enough stem cells for use in the present invention.
[0023] In yet another embodiment, smooth muscle cells may be
derived from peripheral blood. Human blood has circulating adult
progenitor cells that are capable of differentiating into
smooth-muscle-like cells in response to platelet derived growth
factor (PDGF-BB) treatment. (See, e.g., Simper, et al.,
Circulation, 106:1199-1204 (2002)). Thus, in one embodiment of the
present invention, a blood draw is contemplated. Since progenitor
cell populations are present in very low percentages, the cells are
expanded in culture following growth factor-induced differentiation
and selection. Alternatively, the patients may be systemically
treated with agents, such as granulocyte-colony stimulating factor
(G-CSF), granulocyte monocyte colony-stimulating factor (GM-CSF),
or the like, which are known to increase hematopoietic progenitors
in humans by activating and promoting mobilization of these
progenitors from bone marrow into the circulation by several
fold.
[0024] In a next step, step 320 of FIG. 2, cells are isolated from
the harvested tissue. In general, methods of isolation of cells
includes not only harvesting a tissue specimen, but also processing
the specimen so that the cells contained therein are substantially
dissociated into single cells rather than grouped as cell clusters.
Dissociating the cells into single cell components can be
accomplished by any method known in the art; e.g., by mechanical
(filtering) or enzymatic means. Further, in the case of stem cells,
the isolating step includes combining the stem cell-containing
specimen with a cell culture medium comprising factors that (i)
stimulate fibroblast or vascular smooth muscle stem cell growth
without differentiation, and (ii) allow expansion of substantially
only the stem cells. Next, the specimen-medium mixture is cultured
for a few to many cell passages.
[0025] Protocols for the identification of fibroblasts and/or
vascular smooth muscle cells are well established. Markers that can
be monitored and selected for in vascular smooth muscle cells are
smooth muscle cell-specific .alpha. actin (.alpha.SMC), calponin,
smooth muscle myosin heavy chain (SM-MHC) or .beta.-tubulin.
Fibroblasts can be identified morphologically and selected for
vimentin expression. Selection can be accomplished by fluorescence
activated cell sorting (FACS), magnetic activated cell sorting
(MACS), western blotting, or by other techniques known by those
skilled in the art.
[0026] Step 330 shown in FIG. 2 allows for the option of modifying
the fibroblasts and/or vascular smooth muscle cells, such as
genetically altering or engineering the cells or expanding the cell
population in vitro. Methods for genetic engineering or modifying
cells are known to those with skill in the art (see, generally,
Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory
Manual (1982); and DNA Cloning: A Practical Approach, Volumes I and
II (D. N. Glover, ed. 1985)). To genetically engineer the
fibroblasts and/or vascular smooth muscle cells, the fibroblasts
and/or vascular smooth muscle cells may be stably or transiently
transfected or transduced with a nucleic acid of interest using a
plasmid, viral or alternative vector strategy. Nucleic acids of
interest include, but are not limited to, those encoding gene
products that produce or enhance the production of extracellular
matrix components found in fibroblasts and/or vascular smooth
muscle tissue such as cytokines or growth factors, factors that
enhance vascular health and elasticity such as proteolytic
inhibitors, or biological response modulators such as ascorbic acid
(vitamin C) or retinoic acid (vitamin A) that alter the secretory
properties of fibroblasts and vascular smooth muscle cells to
increase collagen type I and III and elastin production. For
example, since tissue repair naturally occurs in an extracellular
matrix environment rich in glycosamines and glycoproteins, it may
make sense to genetically engineer the fibroblasts and/or vascular
smooth muscle cells to produce one or more such compounds.
[0027] Alternatively or in addition, it has been shown that medial
thinning and breakdown of elastin and collagen in the aorta is due,
at least in part, to the effects of matrix metalloproteinases
(MMPs). MMPs (MMP2, gelatinase A; MMP9, gelatinase B, and MMP 12,
metalloelastase) are a group of proteolytic enzymes associated with
the extracellular matrix. MMPs are known to degrade one or more
connective tissue elements and have been implicated in clearing a
path through the extracellular matrix for cell migration. Thus, in
one aspect, the fibroblasts or smooth muscle cells may be
engineered to inhibit the progression of an established
(pre-existing) aneurysm by, e.g., inhibiting MMPs. Suitable
inhibitors may include, for example, endogenous inhibitors, such as
tissue inhibitors of MMPs (TIMPs) and macroglobulins, and synthetic
inhibitors, such as chelating agents (e.g., EDTA and
1,10-phenanthroline), peptides, antibodies, and antibiotics such as
tetracycline and its derivatives.
[0028] The maximal dosage of a bioactive agent such as an MMP
inhibitor in this context is the highest dosage of the bioactive
agent that effectively inhibits elastolytic anti-aneurysmal
activity, but does not cause undesirable or intolerable side
effects. The practitioner is guided by skill and knowledge in the
field, and the present invention includes without limitation
dosages that are effective to achieve the described phenomena.
[0029] The transduction of viral vectors carrying genes for
bioactive compounds into the stem cells can be performed with viral
vectors (adenovirus, retrovirus, adeno-associated virus, or other
viral vectors) that have been isolated and purified. In such
techniques, stem cells are exposed to the virus in serum-free media
in the absence or presence of a cationic detergent for a period of
time sufficient to accomplish the transduction.
[0030] Alternatively, vectors carrying genes for bioactive
compounds can be introduced into the stem cells by use of calcium
phosphate DNA precipitation, cationic detergent methods, liposomes,
TAT-derived cell penetrating peptides, or in three-dimensional
cultures by incorporation of the plasmid DNA vectors directly into
a biocompatible polymer. Perioperative stem cell transfection may
include ultrasound, magnetic field mediated-, or electorporation
techniques. Electroporation protocols are known in the art and also
can be found, e.g., in Maniatis, Fritsch & Sambrook, Molecular
Cloning: A Laboratory Manual (1982); and DNA Cloning: A Practical
Approach, Volumes I and II (D. N. Glover, ed. 1985. For the
tracking and detection of functional proteins encoded by the
introduced genes, the viral or plasmid DNA vectors can contain a
readily detectable marker gene, such as green fluorescent protein
or the beta-galactosidase enzyme, both of which can be tracked by
histochemical means.
[0031] Another method for modifying the stem cells, smooth muscle
cells, and/or fibroblasts prior to delivery to the aneurysmal sac
is to expand the vascular smooth muscle stem cell population.
Basically, the expansion process is accomplished by prolonged in
vitro culturing of the stem cells in the selective cell culture
medium (i.e., the medium that stimulated fibroblast or vascular
smooth muscle stem cell growth without differentiation) from
several to many successive cell passages. On the other hand,
because adipose tissue yields a relatively large number of stem
cells, if adipose tissue is used, in vitro expansion typically is
not necessary.
[0032] Step 340 of FIG. 2 is another optional step that provides
combining the stem cells to be delivered to the blood vessel with a
carrier or scaffold. Many strategies in tissue engineering have
focused on the use of biodegradable polymers as temporary scaffolds
for cell transplantation or tissue induction. The success of a
scaffold-based strategy is highly dependent on the properties of
the material, requiring at a minimum that it be biocompatible, easy
to sterilize, and, preferably, degradable over an appropriate time
scale into products that can be metabolized or excreted. Mechanical
properties are also important in polymer scaffold design for the
regeneration tissues such as connective tissue, adipose tissue or
blood vessels. In addition, scaffold degradation rates should be
optimized to match the rate of tissue regeneration. Ideally
degradable scaffolding polymers should yield soluble, resorbable
products that do not induce an adverse inflammatory response.
Alternatively, biodegradable scaffolds fabricated from naturally
occurring elements, such as collagen, fibrin, hyaluronic acid, with
or without growth factors connected to the backbone can be used as
cell carriers. For general information regarding tissue
engineering, see Ochoa and Vacanti, Ann. N.Y. Acad. Sci., 979:10-26
(2002); Chaikof, et al., Ann. N.Y. Acad. Sci., 961:96-105 (2002);
Griffith, Ann. N.Y. Acad. Sci., 961:83-95 (2002); Weiss, et al.,
U.S. Pat No. 6,143,293; and Zdrahala, et al., U.S. Pat No.
6,376,742.
[0033] In addition to porous scaffolds, the present invention
contemplates using a gel scaffold. Such gels may be synthetic or
semisynthetic gels that may not only stimulate cells through
inclusion of adhesion and/or growth factor moieties, but may also
respond to cells by degrading in the presence of specific cell
cues. In one particularly well-developed family of gels known in
the art, the basic macromer unit is a linear or branched
polyethylene oxide end-capped with chemically reactive groups. Such
a gel is particularly flexible for use in embodiments according to
the present invention as it is intrinsically non-adhesive for cells
and the gel properties can be tailored: the consistency of the gel
can be controlled by the size of the monomers and the gel
thickness; controlled degradation may be had by including
hydrolysable polyester segments or enzyme-cleavable peptides at the
chain ends, and adhesion peptides can be included in the gel at a
concentration to control cell interactions.
[0034] Another type of gel useful in the present invention is a
stimuli-responsive polymer gel. Stimuli-responsive polymer gels are
compounds that can be triggered to undergo a phase-transition, such
as a sol-gel transition. This property aids in reducing the
pressure required to get the polymer-cell suspension through the
delivery means. A preferred system would be a polymer-scaffolding
system that is liquid at room temperature and gels at a temperature
slightly below body temperature.
[0035] As described previously, stem cells respond to soluble
bioactive molecules such as cytokines, growth factors, and
angiogenic factors and can be engineered to secrete such factors as
well as metalloproteinase inhibitors. Thus, these molecules alone
can be used for tissue induction or growth. Alternatively, the
tissue-inductive factors or MMP inhibitors can be incorporated into
the biodegradable polymer of the scaffold, as an alternative to or
in addition to engineering the stem cells to produce such inductive
factors. In yet another alternative, biodegradable microparticles
or nanoparticles loaded with these molecules can be embedded into
the scaffold substrate.
[0036] Alternatively, embodiments of the present invention provide
implanting stem cells along with the differentiation factors
appropriate to induce fibroblast or vascular smooth muscle stem
cell differentiation; however, if such factors are used they are
preferably administered in a time release fashion.
[0037] Thus, the cells can be delivered with or without a cellular
scaffolding or matrix element. However, in addition, the cells and
scaffolding, if present, likely will be delivered in a
pharmaceutically acceptable solution or diluent. For example, the
cells may be delivered in a carrier of sterile water, normal
saline, culture medium or other pharmaceutically acceptable
carrier, alone or in combination with a pharmaceutically acceptable
auxiliary substance, such as a pH adjusting or buffering agent,
tonicity adjusting agent, stabilizer, wetting agent, and the
like.
[0038] Alternate embodiments of this invention include
encapsulating the cells in biodegradable microspheres or capsules,
designed for gradual or measured release over time. Referring again
to FIG. 2, once stem cells have been isolated and/or expanded, they
can be delivered to the blood vessel. To do so, several alternative
delivery means may be employed. For example, syringes or
microneedles may be used to deliver the stem cells directly to the
wall of the blood vessel. Alternatively, catheters or other
apparatus that function to deliver fluids into the walls of blood
vessels may be employed. For example, Jacobsen, et al., U.S. Pat.
No. 6,302,870, describe an apparatus for injecting fluids into the
walls of blood vessels comprising a plurality of laterally-placed
flexible needles disposed within a catheter. Similarly, Linden, et
al., U.S. Pat. No. 5,538,504, describe a drug delivery catheter
that comprises an elongated tubular shaft with a vessel puncturing
element that remains retracted while the catheter is being
deployed, and is then advanced to a protruding position at the site
where the vessel is to be treated. Also, Makower, et al., U.S. Pat.
No. 6,190,353, disclose a similar device for performing drug or
other delivery procedures at specific locations in a blood vessel
using a catheter comprising a deployable element that can penetrate
the wall of the vessel. As another alternative, the cells may be
delivered through a wrap comprised of cells embedded within a
matrix or scaffold, applied either periadventially or
intralumenally. Alternatively, in order to promote the survival of
transplanted cells, chronic cell delivery may be needed. In such an
embodiment, programmable, refillable pumps may be employed to
deliver cells, or cell compositions, that include bioactive agents
to the affected region. As shown by Hartlaub, U.S. Pat. No.
6,348,050, pump-based infusion systems may be used to gradually
monitor cells, together with the appropriate biological response
modulators, to create and maintain the optimal microenvironment for
ensuring cell survival and function.
[0039] FIG. 3 shows a horizontal cross section of an artery. FIG. 3
shows an artery (20), having a tunica intima (26) adjacent to an
internal or inner elastic lamina (28). The tunica media (24) in
FIG. 3 is reduced in size, as compared to a normal artery, due to
degeneration of the smooth muscle and elastic fibers of the tunica
media layer. Encircling the tunica media (24) is the outer or
external elastic lamina (30), the tunica adventitia (22), and the
outer elastic lamina (30). In addition to the cross section of the
catheter (50), an area between the catheter (50) in the lumen (34)
of the artery and the intima layer (26) is present, as well as the
inner lumen of the catheter (56). Also, there is a delivery means
(54) seen in cross section projecting from the catheter (50).
[0040] In some procedures where vessel repair is necessarily
extensive-particularly at aneurismal sites--a stent graft may be
used, and a catheter may be tracked along side the stent to deliver
the stem cells to the media of the vessel. Such stents are known in
the art. In some cases, it may be preferable to use a biodegradable
stent. Such devices are disclosed, for example in Jadhav, U.S. Pat.
No. 6,368,346; and Healy, et al., U.S. Pat. No. 5,670,161. in such
cases, where the discease is an an advanced stage and a stent graft
is employed, cells with or without scaffolds, gels or encapsulation
may be injected directly into the aneurysmal sac region for the
purpose of remodeling and/or reinforcement.
[0041] FIG. 4 shows one embodiment of delivery of the stem cells,
smooth muscle cells, or fibroblasts involving the transluminal
placement of a prosthetic arterial stent graft 10 positioned in an
aorta 12. The stent spans, within the aorta 12, an aneurysmal
portion 14 of the aorta 12. The aneurysmal portion 14 is formed due
to a bulging of the aorta wall 04. As a result, an aneurysmal sac
18 is formed of distended vessel wall tissue. The stent graft 10 is
positioned spanning the sac 18 providing both a passageway for
blood flow through the aorta 12 and sealing of the aneurysmal
portion 14 of the aorta 12 from additional blood flow from the
aorta 12. In addition, FIG. 4 shows a portion of a catheter 30
tracked along side of the stent graft 10. The catheter 30 has a
distal end 32 that resides in the aneurysmal portion 14 of the
aorta 12. The cells 60 are delivered to the aneurysmal site through
the distal end 52 of the catheter 50. As discussed, the cells
delivered may or may not be bioengineered, and may or may not be
accompanied by cellular scaffolding, delivery solutions and/or
soluble bioactive molecules such as cytokines, growth factors, and
angiogenic factors. The cells and other elements, if present,
support or bolster the aneurysm, while providing the factors
necessary to stimulate the growth of new tissue to continue to
support the aneurysm.
EXAMPLES
Cultivation of Smooth Muscle Progenitor Cells
[0042] Smooth muscle progenitor cells may be isolated from human
blood, as demonstrated by Simper, et al. (see, Circulation,
106:1199-1204 (2002)). To do so, human mononuclear cells are
isolated from peripheral buffy coat blood, suspended in endothelial
growth medium (EGM-2) and seeded on collagen type I matrix. The
seeded smooth muscle progenitor cells were then grown in
platelet-derived growth factor BB-enriched medium to promote smooth
muscle outgrowth and expansion. Evaluations of cell phenotype may
be carried out by western blotting or FACS sorting using monoclonal
antibodies to .alpha.SMA, human smooth muscle myosin heavy chain,
calponin and Flt1, CD 31, VE cadherein, vWF, CD34, Tie-2 and Flk1
receptors. Smooth muscle outgrowth cells are positive for smooth
muscle cell-specific .alpha. actin (.alpha.SMA), myosin heavy
chain, calponin, and CD34, Flt1 and Flk1 receptors. CD31, VE
cadherein, vWF, and Tie-2 serve to identify contaminating
(non-vascular smooth muscle) cell types.
Cultivation of Fibroblasts
[0043] Fibroblasts may be isolated from human dermal tissue, as
demonstrated by Tonello, et al. (see, Biomaterials
24:1205-1211(2003)). In this protocol, following epithelial sheet
dispase removal, the dermis is cut into small pieces (2-3 mm2) and
fibroblasts are isolated by sequential trypsin and collagenase
digestion. The cells are then cultured with DMEM medium
supplemented with 10% fetal bovine serum plus L-glutamine (2 mM)
and penicillin(100 U/ml)/streptomycin(100 .mu.g/ml). The medium is
changed every three days and the cells are harvested by trypsin
treatment.
Differentiation Protocol
[0044] For the development of stem cells into differentiated
vascular smooth muscle cells, the stem cells may be cultivated in
3-dimensional aggregates called embryoid bodies (EBs) by the
hanging drop method, by mass culture, or by differentiation in
methylcellulose. Here, the hanging drop method is described.
[0045] 1. Prepare a cell suspension containing a defined ES cell
number of 800 cells in 20 mL of differentiation medium (DMEM (Gibco
cat. No. 42200-030) supplemented with 20% FCS and, for each 100 mL
medium, 1 mL 200 mM L-glutamine, 1 mL .beta.-mercaptoethanol, 1 mL
of nonessential amino acids stock (100.times., Gibco cat. No.
25030-024)).
[0046] 2. Place 20 mL drops (n=50-60) of the ES cell suspension on
the lids of 100-mm bacteriological Petri dishes containing 10 mL
PBS.
[0047] 3. Cultivate the ES cells in hanging drops for 2 days. The
cells will aggregate and form one EB per drop.
[0048] 4. Rinse the aggregates carefully from the lids with 2 mL of
medium, transfer into a 60-mm bacteriological Petri dish with 5 mL
of differentiation medium, and continue cultivation in suspension
for 7 days until the time of plating.
[0049] 5. Plate EBs at day 7 and induce differentiation of vascular
smooth muscle cells by treatment with 10.sup.-8 M retinoic acid (in
ethanol or DMSO) and 0.5.times.10.sup.-3 M db-cAMP between day 7
and 11 after plating (duration and treatment time has to be
optimized for each cell line). The first spontaneously contracting
vascular smooth muscle cells, which express the vascular-specific
splice variant of the vascular smooth muscle myosin heavy chain
(MHC) gene, appear in the EBs around 1 week after plating. Change
the medium during the differentiation period every day or every
second day.
[0050] 6. A similar vascular smooth muscle cell induction is
achieved by cultivating stem cells (n=600) as EBs in hanging drops
in differentiation medium II (DMEM supplemented with 15%
dextran-coated charcoal-treated FCS, and, for each 100 mL medium, 1
mL 200 mM L-glutamine, 1 mL .beta.-mercaptoethanol, 1 mL of
nonessential amino acids stock (100.times., Gibco cat. No.
25030-024), 10 .mu.L of a 3.times.10.sup.-4 M stock solution of
Na-selenite, 2.5 mL of 7.5% stock solution bovine serum albumin,
and 0.25 mL stock solution (4 mg/mL) transferrin) containing 2
mg/mL transforming growth factor .beta.1 from day 0 to 5. Plate the
EBs at day 5. The first spontaneously contracting vascular smooth
muscle cells appear in EBs 10 days after plating, and maximal
vascular smooth muscle cell differentiation (60%) is achieved at
day 5 plus 24 hours to day 5 plus 28 hours.
[0051] 7. Alternatively, ES cells (n=400) may be differentiated in
hanging drops in M15 (DMEM plus 15% FCS, 0.1 mM
beta-mercaptoethanol, 2 mM L-glutamine, 0.05 mg/mL streptomycin,
and 0.03 mg/mL penicillin). After plating at day 4.5, the medium is
partially exchanged every third day. Maximal vascular smooth muscle
cell differentiation (30%) is achieved at day 4.5 plus 17 hours to
day 4.5+19 hours.
[0052] 8. For morphological analysis, transfer a single EB into
each well of gelatin (0.1%)-coated microwell plates, transfer 20 to
40 EBs per dish onto 60-mm tissue culture dishes containing 4
coverslips (10.times.10 mm) for immunofluorescence, or transfer 15
to 20 EBs onto 60-mm tissue culture dishes for reverse
transcription PCR (RT-PCR) analysis of EB outgrowths.
[0053] While the present invention has been described with
reference to specific embodiments, it should be understood by those
skilled in the art that various changes may be made and equivalents
may be substituted without departing from the true spirit and scope
of the invention. In addition, many modifications may be made to
adapt a particular situation, material, or process to the
objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the
invention.
[0054] All references cited herein are to aid in the understanding
of the invention, and are incorporated in their entireties for all
purposes.
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