U.S. patent application number 13/523334 was filed with the patent office on 2013-05-16 for ventricular assist device capable of implantation of stem cells.
This patent application is currently assigned to UNIVERSITY OF UTAH FOUNDATION. The applicant listed for this patent is David A. Bull, Rafe C. Connors, Harold M. Erickson, Sung Wan Kim, James Yockman. Invention is credited to David A. Bull, Rafe C. Connors, Harold M. Erickson, Sung Wan Kim, James Yockman.
Application Number | 20130123624 13/523334 |
Document ID | / |
Family ID | 39111532 |
Filed Date | 2013-05-16 |
United States Patent
Application |
20130123624 |
Kind Code |
A1 |
Bull; David A. ; et
al. |
May 16, 2013 |
VENTRICULAR ASSIST DEVICE CAPABLE OF IMPLANTATION OF STEM CELLS
Abstract
A biologic ventricular assist device that also has the
capability to capture, grow, and administer stem cells to
regenerate and restore damaged myocardium in the heart. The device
works in conjunction with a traditional ventricular assist device
and possesses an additional external path or tube that is in-line
with the path of the ventricular assist device. The external path
allows for the administration of stem cells, genes, genetically
modified cells or other therapeutic biologic or pharmacologic
agents, as well as leading to a stem cell collecting accessory that
captures circulating stem cells. The stem cell collecting accessory
is also associated with a chamber for culturing the captured stem
cells. The cultured stem cells can be delivered back to the heart
by an electro-mechanical or ultrasound/echocardiographic delivery
system that runs through the external path back into the
ventricular assist device and allows for the delivery of the stem
cells, or other therapeutic biologic or pharmacologic agents,
directly into the internal chambers of the heart. Administering the
stem cells, genes, genetically modified cells or other therapeutic
biologic or pharmacologic agents, either alone or in combination,
to the heart allows the myocardium to regenerate and repair itself
even while the heart is attached to the ventricular assist device,
ultimately allowing the heart to regenerate, recover and allow the
VAD to be removed.
Inventors: |
Bull; David A.; (Salt Lake
City, UT) ; Connors; Rafe C.; (Keswick, VA) ;
Erickson; Harold M.; (Cora, WY) ; Yockman; James;
(West Jordan, UT) ; Kim; Sung Wan; (Salt Lake
City, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bull; David A.
Connors; Rafe C.
Erickson; Harold M.
Yockman; James
Kim; Sung Wan |
Salt Lake City
Keswick
Cora
West Jordan
Salt Lake City |
UT
VA
WY
UT
UT |
US
US
US
US
US |
|
|
Assignee: |
UNIVERSITY OF UTAH
FOUNDATION
Salt Lake City
UT
|
Family ID: |
39111532 |
Appl. No.: |
13/523334 |
Filed: |
June 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12513310 |
Feb 5, 2010 |
8241198 |
|
|
PCT/US2007/023251 |
Nov 5, 2007 |
|
|
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13523334 |
|
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60856562 |
Nov 3, 2006 |
|
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Current U.S.
Class: |
600/439 ;
604/522 |
Current CPC
Class: |
A61M 1/1006 20140204;
A61M 1/122 20140204; A61M 1/3472 20130101; A61M 1/3621 20130101;
A61M 1/1001 20140204; A61M 1/3667 20140204; A61M 1/1008 20140204;
A61M 1/12 20130101; A61M 1/3618 20140204; A61M 1/127 20130101; C12N
5/0662 20130101; A61K 35/34 20130101; A61B 8/12 20130101; A61M
1/3689 20140204 |
Class at
Publication: |
600/439 ;
604/522 |
International
Class: |
A61K 35/34 20060101
A61K035/34; C12N 5/071 20060101 C12N005/071 |
Claims
1-5. (canceled)
6. A method for delivering cardiac s e cells or cardiac progenitor
cells to a heart, comprising: a) isolating cardiac stem cells or
cardiac progenitor cells during surgery to give isolated cardiac
stem cells or progenitor cells; b) culturing the isolated cardiac
stem cells or cardiac progenitor cells to give cultured cardiac
stem cells or cardiac progenitor cells; and c) reintroducing the
cultured cardiac stem cells or progenitor cells to the heart.
7. The method of claim 6. wherein the cardiac stem cells or cardiac
progenitor cells are isolated at the time a ventricular assist
device (VAD) is placed.
8. The method of claim 6, wherein the cardiac stem cells or cardiac
progenitor cells are isolated using streptavidin-coated magnetic
beads.
9. The method of claim 6, wherein the cardiac stem cells or cardiac
progenitor cells are isolated from a sample excised from the left
ventricular apex.
10. The method of claim 6, wherein the cardiac stem cells or
progenitor cells are grown to confluence inside the body.
11. The method of claim 6. wherein the cardiac stem cells or
cardiac progenitor cells are grown to confluence outside the
body.
12. The method of claim 6, wherein the cardiac stem cells or
cardiac progenitor cells are characterized via flow cytometery for
phenotypic surface markers to ensure efficient isolation.
13. The method of claim 6. wherein the cardiac stem cells or
cardiac progenitor cells are reintroduced to the heart using an
electro-mechanical delivery system, an ultrasound/echocardiographic
imaging delivery system. or a combination of both.
14. A method of repairing or restoring a damaged heart in a subject
comprising: a) capturing circulating stem cells in the subject to
give captured stem cells; b) growing captured stem cells to a
desired mass using a bio-reactor within an in-line chamber of a
biologic ventricular assist device to give grown captured stem
cells; c) returning the grown captured stem cells to the damaged
heart.
15. A method of repairing or restoring a damaged heart in a subject
comprising: a) capturing circulating stem cells in the subject to
give captured stem cells; b) growing captured stem cells to a
desired mass outside of the subject to give grown captured stem
cells; c) returning the grown captured stem cells to the damaged
heart.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/856,562, filed on Nov. 3, 2006, entitled
VENTRICULAR ASSIST DEVICE CAPABLE OF IMPLANTATION OF STEM CELLS,
the entire content of which is hereby incorporated by
reference.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] No federal funds were used in the development of the present
invention.
BACKGROUND
[0003] This invention pertains to ventricular assist devices, and
particularly to a ventricular assist device that can support a
heart either through culture and/or therapeutic external
administration of stem cells.
[0004] Aging of the population and prolongation of the lives of
cardiac patients by modern therapeutic innovations has led to an
increasing prevalence of heart failure ("HF"). Despite improvements
in both medical and surgical therapy, the mortality rate in
patients with HF has remained unacceptably high.
[0005] The surgical management of patients with end-stage heart
failure is slowly evolving. Heart transplantation remains the
ultimate treatment for heart failure, but the persistent shortage
of donor hearts continues to limit the annual growth of this
approach. Thus, heart transplantation is not an available option
for most patients with HF and continues to be performed only at
large, highly specialized medical centers. Ventricular assist
devices ("VADs") are currently most commonly used as a bridge to
transplantation, but are now being designed as destination therapy
for many HF patients.
[0006] Studies have been completed showing the beneficial effect of
gene therapy for myocardial neovascularization. Animal studies show
evidence of cardiac progenitor cells, or cardiac stem cells,
existing in the atria and ventricles. These cells have been
harvested from myocardium of several different vertebrate species
and subsequently grown in vitro. These same types of cells exist in
human myocardium.
[0007] The current state of the art therapy is in evolution. VADs
are coming to the forefront of therapy with cardiac
transplantation. However, at the present time, VADs can only
support a patient in cardiogenic shock until a donor heart becomes
available for transplant. The current generation of VADs do nothing
to help regenerate the heart to restore it to a normal level of
functioning. Indeed, research demonstrates that the longer a
ventricular assist device is in place, the more likely it is that
the heart muscle will be replaced by scar tissue, resulting in an
atrophic, non-functioning heart, incapable of functioning without
the VAD in place. In addition, a few centers have gained FDA
approval to begin Phase I human clinical trials with bone marrow
mononuclear cells as therapy for myocardial ischemia. While bone
marrow mononuclear cell therapy holds significant promise for
therapy, early long-term data indicates that the bone
marrow-derived cells do not differentiate into mature myocytes or
blood vessels.
[0008] What is needed, therefore, is a therapy for HF patients that
can support a patient in end-stage heart failure, such as a VAD,
and that can utilize other biologic and/or pharmacologic therapies
to regenerate the heart and help restore it to a normal level of
functioning.
SUMMARY
[0009] The present invention relates generally to the field of
ventricular assist devices. In particular, this invention relates
to a biologic ventricular assist device that also has the
capability to capture, grow, and administer stem cells, other
therapeutic biologic agents and other therapeutic pharmacologic
agents, either alone or in combination, to regenerate and restore
damaged myocardium in the heart.
[0010] Native progenitor or stem cells which are capable of
repairing and regenerating the organs of the human body offer a
novel means to address the problem of myocardial atrophy with the
VAD in place. These progenitor or stem cells are routinely present
both within solid organs and circulating in the blood stream. The
advantage of the cardiac progenitor cells is that they are already
resident within the myocardium and have demonstrated in other
animal studies to differentiate only into cardiac myocytes,
coronary arterioles, and capillary structures, and are already
believed to do so within the myocardium during ischemic periods.
Unfortunately, their numbers at any given time are so small that
these cells have not been thought to be a practical means of
externally directing large scale tissue regeneration or repair. The
current biologic ventricular assist device allows for the capture,
growth, and administration of therapeutic biologic or pharmacologic
entities which include but are not limited to: cells, stem cells,
genes, genetically modified and/or cultured stem cells, drugs, and
components of the extracellular matrix either alone or in
combination, to allow them to be applied in a truly therapeutic
fashion to regenerate and restore damaged myocardium.
[0011] The biologic ventricular assist device, BIOVAD.TM. (any
ventricular assist device that allows for the capture, growth, and
administration of therapeutic biologic or pharmacologic entities
including but not limited to: cells, stem cells, genes, genetically
modified and/or cultured stem cells, drugs, and components of the
extracellular matrix either alone or in combination), offers a
novel means to regenerate and restore the native heart while the
VAD is in place, with the ultimate goal of allowing the removal of
the VAD and obviating the need for a heart transplant. The biologic
ventricular assist device does this by using the native cardiac
progenitor cells isolated at the time that the VAD is placed,
growing them to confluence either within or outside the body, then
re-administering them to the patient via an electro-mechanical
and/or ultrasound/echocardiographic imaging delivery system which
allows electro-mechanical echocardiographic imaging of the heart,
such as a NOGA catheter system. This electro-mechanical and/or
ultrasound/echocardiographic imaging and delivery system will pass
through an external sleeve system placed along the drive line and
along the course of the device back into the internal cardiac
chambers to allow the delivery of the appropriate dose of cardiac
progenitor or stem cells. This external to internal sleeve system
allows for repeated delivery of therapeutic biologic or
pharmacologic entities including but not limited to: cells, stem
cells, genes, genetically modified and/or cultured stem cells,
drugs, and components of the extracellular matrix, either alone or
in combination, to the native myocardium over time, allowing the
heart to repair itself in a graded, step-wise, physiologic
fashion.
[0012] The tissue obtained during VAD placement is usually
discarded following surgery. During cannulation of the atrium prior
to going on cardiopulmonary bypass, a small and inconsequential
piece of atrium can be obtained from the cannulation site. Further,
the ventricular apex is cored out for placement of the device. This
section of the wall of the apex of the left ventricle and/or the
resected portion of the atrium are used as the source of cardiac
progenitor and stem cells resident in the myocardium. These cells
can be isolated and grown externally to supply the cardiac stem
cells for later re-administration.
[0013] The biologic ventricular assist device also utilizes an
in-line chamber to capture circulating stem cells which are
normally in small numbers in circulation, grow them up to a
critical mass or density at which they become therapeutic using a
"bio-reactor" within the chamber, and then return them to the
damaged heart either in an internal automated or external
selectively determined fashion to repair and restore the damaged
heart. This ultimately allows for the removal of the ventricular
assist device. The principle of the biologic ventricular assist
device is that a chamber is placed in-line with the circuit through
which blood flows. This in-line chamber contains a series of
polymeric filters embedded with chemokines and cytokines which
serve to attract and capture stem cells which are circulating in
very low numbers in the blood stream. The chamber also contains
nutrient elements which allow the stem cells to proliferate in a
contained "bio-reactor." Once the cells reach confluence, they can
be removed to allow genetic modification prior to re-administration
or they can be returned directly back to the heart to help
regenerate viable heart tissue and ultimately restore a heart which
is capable of supporting its own function sufficiently to allow the
VAD to be removed.
[0014] The chamber to capture circulating stem cells and allow
their proliferation within a bioreactor is adaptable to any in-line
blood circuit with any connection to the bloodstream. The most
obvious related extensions of this technology would be to patients
undergoing dialysis, plasmapheresis, or any clinical setting in
which a chamber system can be placed in-line to capture circulating
cellular elements within the blood.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a schematic of one embodiment of the biologic
ventricular assist device.
[0016] FIG. 2 shows an schematic of one embodiment of the stem cell
collection accessory.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0017] One aspect of the present invention pertains to a biologic
ventricular assist device (FIG. 1) that is capable of capturing,
culturing, and delivering stem cells within a heart to which the
device is attached. The ventricular assist device includes an
inflow path (7,8), a pump (11), and an outflow path (6,2). The
inflow path (7,8) of the ventricular assist device is attached to
the left ventricle of the heart (3), and blood flows into the
inflow path (7,8) from the left ventricle (3). The inflow path
(7,8) then passes into the pump (11), which directs the blood into
the outflow path (6,2). The outflow path directs the blood back
into the ascending aorta of the heart (1). A drive line (16)
typically connects the pump to a drive unit (18) that is external
to the body. In the current invention, an external path is also
attached at various points to the inflow path (7,8), the pump (11),
and the outflow path (6,2) of the ventricular assist device (FIG.
1). Blood also flows through the external path. The external path
leads to a stem cell collection accessory ("SCCA", FIG. 2) which
captures circulating stem cells in the blood or from the heart.
[0018] Another aspect of the present invention is the stem cell
collection accessory ("SCCA", FIG. 2), which is a path or chamber
through which blood flows after it is directed there by the
external path. The path or chamber has walls of selective
permeability and one or more layers of gels or polymers (36, 35,
33, 31, 34) having a chemical gradient sufficient to cause
migration of the stem cells from the blood through the walls and
into the surrounding gel.
[0019] In the present invention, once the captured stem cells are
grown to confluence, the stem cells are re-suspended and delivered
back to the internal chambers of the heart using a delivery system
that also passes through the external path. This external path
leading back to the heart allows for repeated delivery of not only
the captured and cultured stem cells but also therapeutic biologic
or pharmacologic entities including but not limited to: cells, stem
cells, genes, genetically modified and/or cultured stem cells and
drugs, either alone or in combination, over time.
[0020] In a preferred embodiment, an inflow cannula (7) and inflow
valve conduit (8) passing out of the left ventricle of the heart
(3) make up the inflow path of the biologic ventricular assist
device (FIG. 1). The inflow path (7,8) directs the blood into the
pump (11), which then directs the blood into the outflow path
(6,2). In a preferred embodiment, an outflow valve conduit (6) and
outflow graft (2) make up the outflow path. In an additional
preferred embodiment, the external path of the biologic ventricular
assist device is made up of a left ventricle tube (9), an aortic
tube (10), a percutaneous tube (20), and a return tube (12).
[0021] FIG. 1 shows one preferred embodiment of the biologic
ventricular assist device. The heart is illustrated, including the
left ventricle (3) and aorta (1). Positioned at the ventricular
apex (4) is an apical sewing ring (5) that allows attachment of the
inflow cannula (7) of the device. The inflow cannula (7) passes
into the inflow valve conduit (8), allowing blood exiting the left
ventricle (3) to flow into the pump (11). Also exiting the inflow
valve conduit (8) is a left ventricle tube (9) through which blood
can bypass the pump (11) and proceed in a direction toward the stem
cell collection accessory ("SCCA", FIG. 2). The left ventricle tube
(9) can also contain one or more accessory sleeves (23) or lines
for instrumentation (26). In a preferred embodiment, these
accessory sleeves can be called left ventricle accessory sleeves
(23). The tube may be attached at the inflow valve conduit (8) with
one-way valves for the accessory sleeves (23) and open ports for
the lines involved with blood flow.
[0022] At a branch point (27), the left ventricle tube (9) merges
into a percutaneous tube (20) leading out of the body, past the
incision and out of the skin, with a drive line (16) leading
eventually back to the drive unit (18). Also at this branch point
(27), an aortic tube (10) enters the percutaneous tube (20) from a
point at the outflow valve conduit (6). The aortic tube (10) may
contain one or more accessory sleeves (22) or lines allowing for
the bypass flow of blood directly out of the outflow valve conduit
and the aorta (21) toward the stem cell collection accessory
("SCCA"). In a preferred embodiment, these may be called an aortic
accessory sleeve (22) and a bypass flow line (21). The aortic tube
(10) may be attached at the outflow valve conduit (6) with one-way
valves for the accessory sleeves and open ports for the lines
involved with bypass blood flow. The percutaneous tube (20) also
contains the accessory sleeves and lines allowing for ex-vivo
delivery of therapeutic biologic or pharmacologic entities
including but not limited to: cells, stem cells, genes, genetically
modified and/or cultured stem cells, drugs, and components of the
extracellular matrix, either alone or in combination, within the
other tubes, as well as the coaxial drive line that runs between
the drive unit (18) and the pump (11). Blood exiting the pump (11)
that is not involved in bypass flow passes through the outflow
valve conduit (6) and through the outflow graft back (2) into the
ascending aorta (1).
[0023] Also entering the percutaneous tube (20) at the branch point
(27) is a return tube (12) that can return blood to the pump (11)
after it passes out of the stem cell collection accessory ("SCCA",
FIG. 2) and through the percutaneous tube (20). The return tube
(12) can also contain one or more accessory sleeves (23, 26)or
lines allowing for ex-vivo delivery of therapeutic biologic or
pharmacologic entities including but not limited to: cells, stem
cells, genes, genetically modified and/or cultured stem cells,
drugs, and components of the extracellular matrix, either alone or
in combination, as well as accessory sleeves or lines allowing for
instrumentation. Where the return tube (12) meets the pump (11), a
drive line (16) may also enter the tube for passage back to the
drive unit (18). These accessory sleeves or lines may be called in
a preferred embodiment left ventricle accessory sleeves (23), and a
return flow line (25).
[0024] The percutaneous tube (20) passes outside of the body at the
skin line and enters an adapter containing a vent filter, a stem
cell collection accessory ("SCCA", FIG. 2), and one-way access
valves for access to the aortic accessory sleeve, the left
ventricle accessory sleeve, and the second left ventricle accessory
sleeve (13). The drive line (16) can continue past the adapter to
the drive unit (18).
[0025] FIG. 2 shows an illustration of a preferred embodiment of a
stem cell collecting accessory. Blood enters the stem cell
collecting accessory from the bypass flow line (37). The bypass
flow line (37) contains blood that passed through the pump (11),
exited at the outflow valve conduit (6), passed through the aortic
tube (10), passed into the percutaneous tube (20) at the branch
point (27), and entered the adapter. This blood flow comes from the
high pressure side of the device. The stem cell collecting
accessory (FIG. 2) is generally surrounded by a biocompatible
polymer (36). Within the stem cell collecting accessory itself is a
chamber (39) through which blood passes. O-rings (42) may be
located at either end of the chamber. The first layer surrounding
the chamber is a cell-permeable membrane (35). The O-rings (42)
also serve as formation aids for this cell-permeable membrane.
Outside of the cell-permeable membrane (35) is an inner
enzyme-degradable thermoreversible hydrogel (34) which contains a
gradient of cytokines diffusing toward the flow of blood. The
gradient in this hydrogel serves to capture circulating progenitor
or stem cells as they migrate through the cell-permeable membrane.
Chemoinvasive cells (30) migrate into this hydrogel from the blood.
Outside the hydrogel is a cytokine-permeable membrane(33) through
which the stem cells do not easily pass. Outside of the
cytokine-permeable membrane (33) is an outer enzyme-degradable
thermoreversible hydrogel (31) that is doped with cytokines in
sufficient concentration to sustain an approximately unchanging
gradient over the exposure lifetime. This outer hydrogel is
moderately diffusion-inhibiting. The outermost layer is a rigid
outer wall (32).
[0026] The blood that flows through the stem cell collecting
accessory (FIG. 2) then enters the return flow line (25). The
return flow line (25) passes through the percutaneous tube (20),
passes into the return tube (12) or any line allowing for the
return flow of blood at the branch point (27), and re-enters the
pump (11). This blood flow is directed to the low pressure side of
the device.
[0027] Once they have grown to confluence within the stem cell
collection assembly, the cardiac or circulating progenitor or stem
cells are removed from the stem cell collecting accessory,
re-suspended in solution and then re-administered via the
electro-mechanical and/or ultrasound/echocardiographic imaging and
delivery system directed through the external sleeve system within
the percutaneous tube and other tubes placed along the drive line
and along the course of the device back into the internal cardiac
chambers to allow the delivery of the appropriate dose of cardiac
progenitor or stem cells.
Example 1
Cardiac Progenitor Cell Isolation
[0028] Isolation and Characterization of Cardiac Stem Cells. Tissue
samples are obtained from patients receiving a left ventricular
assist device (LVAD). The 1-2 cm.sup.3 samples are excised from the
left ventricular apex to allow for placement of the device.
Typically, this "core" is discarded upon excision. However, this is
a viable source of tissue, regardless of the pathological
background, to isolate resident cardiac stem cells.
[0029] Processing of Human Cardiac Stem Cells from Clinical
Samples. The cardiac tissue "core" is minced with a scalpel into
2-3 mm.sup.3 pieces, and 20 pieces (generally, 500 mg) are placed
into 2 ml of 0.13 mg/ml Liberase Blendzyme 4 (Roche Diagnostics
Corp., San Diego, Calif.) re-suspended in serum free Hams F12
media. The tissues are incubated for 30 minutes with a brief
vortexing every 10 minutes. The larger tissues are collected by
centrifugation at 500 RPM for 2 minutes and the supernatant
collected and strained through a 30 uM nylon mesh. The remaining
tissue is re-suspended in 2 ml of 0.13 mg/ml Liberase Blendzyme 4
and the procedure is repeated for a total of three times. Each time
the supernatant is collected, the sample is strained through the
nylon mesh and the cells spun at 800 RPM for 10 minutes to pellet
the cells. These cells are re-suspended in calcium and magnesium
free PBS supplemented with 0.1% BSA (Sigma, St. Louis, Mo.) and 2
mM EDTA and placed in the incubator until all sample digestions
have been completed. Following digestion, the cells will be pooled
and counted on a hemacytometer. Viability will be measured by
trypan blue exclusion.
[0030] Magnetic isolation of CD117.sup.pos/Pgp.sup.pos stem cells.
CD117.sup.pos/Pgp.sup.pos (P-glycoprotein) cells are labeled with 1
.mu.g of biotinylated mouse anti-human CD 117 (eBioscience, San
Diego, Calif.) and biotinylated mouse anti-Pgp (Chemicon, Temecula,
Calif.) per 1.times.10.sup.7 cells for 30 minutes at 4.degree. C.
Following incubation, the cells are washed in PBS plus 0.1% BSA and
2 mM EDTA and re-suspended in 2.times.10.sup.7 cells/ml of wash
buffer. A total of 25 .mu.l of Streptavidin coated CELLection
Dynabeads.RTM. (Dynal.RTM., Invitrogen, Carlsbad, Calif.) is added
to the cells and incubated with gentle tilting and rotation for 30
minutes. The cells are placed into the Dynal.RTM. MPC-L magnet for
2 minutes. The supernatant is removed and the bead bound cells are
washed 3 times in wash buffer. The supernatant is collected and
stored separately. The bead bound cells are re-suspended in 200
.mu.l of RPMI 1640 plus 1% FBS and 4 .mu.l of 10,000U/ml DNaseI is
added for 15 minutes with gentle tilting and rotation. The sample
is then vortexed vigorously and placed into the magnet for 2
minutes. The supernatant is then collected and the tube washed once
in RPMI 1640 plus 1% FBS. The supernatant is pelleted at 800 RPM
for 10 minutes and re-suspended in growth media, Ham's F12
supplemented with 5% FBS and 10 ng/ml each of LIF (Chemicon) and
bFGF (Chemicon). The cells are counted on a hemacytometer and
plated in a 6-well plate (Nunc, Rochester, N.Y.) at
2.times.10.sup.4 cells/cm.sup.2. The supernatant is replaced after
one week and the plate washed with PBS and maintenance media is
added, Ham's F12 supplemented with 5% FBS, 10 ng/ml LIF and bFGF
and 20 ng/ml of EGF (Chemicon). Media is changed every 3-4 days
until 50% confluency. Upon 50% confluency, the plate is passaged
into a 75 cm.sup.2 flask (Nunc). The negatively sorted cells are
plated at a density of 5.times.10.sup.5cells/cm.sup.2 on 75
cm.sup.2 flasks in growth media (described in D.1.2.). These cells
are treated similarly to positive selected cells in regards to
media and passaging. Yield, morphology, homogeneity, and cell
growth characteristics are documented for each sample and their
isolates.
[0031] Adherent isolation of cardiac stem cells. Cells processed
from clinical cardiac samples are plated at a density of
5.times.10.sup.5 cells/cm.sup.2 on 75 cm.sup.2 flasks in growth
media. Adherent and non-adherent fractions are collected based on
the following time points: 1 hour, 2 days, 5 days and 7 days. The
adherent fractions then have the media replaced with maintenance
media. The supernatant containing the non-adherent cells, is
pelleted and re-suspended in maintenance media and plated on 75
cm.sup.2 flasks. Once 50% confluence is reached, the cells are
passaged to 175 cm.sup.2 flasks in maintenance media. Yield,
morphology, homogeneity, and cell growth characteristics are
documented for each sample and their isolates.
[0032] Flow cytometry characterization. Cells are characterized
through flow cytometry for phenotypic surface markers to determine
the efficacy and homogeneity of the isolation techniques. Cells are
stained, with mouse anti-human antibodies (Pharrningen, BD
Biosciences, Mississauga, Canada) for stem cell markers CD105,
CD117, CD133, CD166, the drug resistance marker, P-glycoprotein
(Pgp), as well as lineage markers, CD4, CD8, CD20, CD34, CD45,
CD45RO and the endothelial marker CD31 and adhesion marker CD44.
Cells are trypsinized with TrypLE (Invitrogen), pelleted, and
re-suspended in PBS plus 5% BSA at a density of 1.times.10.sup.6
cells/ml. 200 .mu.l of the cells are aliquoted into 12.times.75 mm
tubes and 0.5 mg of appropriate antibodies are added to each tube.
Four different antibodies are added per tube that possess
particular fluorescent characteristics so that there is little
fluorescent emission overlap. The antibodies are incubated at
4.degree. C. for 30 minutes, washed in PBS plus 5% BSA and
re-suspended in 1 ml PBS. Cells are analyzed using the Becton
Dickinson FACScan Analyzer and CellQuest software
(Becton-Dickinson, BD Biosciences).
[0033] Differentiation capacity of cardiac stem cells. Cells are
trypsinized and placed onto a Nunc eight-well LabTek.TM. chamber
slides (Sigma) at 1.times.10.sup.3 cells/cm.sup.2 and grown under
normal or differentiative conditions. Media are changed every 3-4
days. The number of positive cells are counted using a fluorescent
microscope and representative micrographs are taken with the
Olympus BX5OWI (Center Valley, Pa.) two photon confocal microscope
available. Background staining consists of Prolong Gold.TM.
anti-fade plus DAPI (Molecular Probes, Invitrogen).
[0034] Cardiomyogenic differentiation of stem cells following
co-culture with neonatal cardiomyoeytes. To induce cardiomyogenic
differentiation, human cardiac stem cells ("hCSC's") are
co-cultured with neonatal human ventricular myocytes ("NRVMs").
CSCs are labeled with PKH-26 (Sigma) prior to addition to the NRVMs
cultures at a 1:4 ratio and cultured for up to 2 weeks with media
changes every 3-4 days. PKH-26 labeled cells retain both biological
and proliferative activity, and are ideal for cell tracking
studies. The linkers are physiologically stable (lasting up to 100
days) and show little to no toxic side effects. PKH-26 has an
excitation and emission of 551/567 nm that is compatible with
rhodamine or phycoerythrin detection systems. However, it may also
be excited by the 488 nm emission of an argon-ion laser. Briefly,
cells are trypsinized from the plate, pelleted and washed twice in
serum-free media. After the final wash, the cells are suspended at
4.times.10.sup.5 in 50 .mu.l diluent. 50 .mu.l of 2.times. PKH-26
dye is added and the cells are incubated at room temperature for
approximately 5 minutes. This time may change as each cell type
exhibits different properties in lipid uptake. To ensure homogenous
staining, cells are incubated for different times and analyzed by
confocal microscopy. The reaction is stopped by adding an equal
amount of growth media with FBS and the cells are washed 3-5 times
to remove any unbound dye. Cells are stained for rabbit anti-human
cardiac troponin I (Abcam, Cambridge, UK), biotinylated goat
anti-human GATA-4 and mouse anti-human Nkx2.5 (R&DSystems,
Minneapolis, Minn.).
[0035] Endothelial differentiation of stem cells. To induce
differentiation into endothelial cells, hCSCs are plated at
5.times.10.sup.4/cm.sup.2 in DMEM or EBM-2 (Cambrex) with 2% FBS,
supplemented with 10.sup.-8 M dexamethasone and 10 ng/ml VEGF, in
chamber slides coated with either 0.1% gelatin or fibronectin for
14 days with media changes every 3-4 days. Tube-like structures may
form after five days, but after 14 days they exhibit endothelial
specific markers. Cells are stained for rabbit anti-human von
Willebrands Factor ("vWF"), and mouse anti-human CD31.
[0036] Smooth muscle differentiation. Cardiac stem cells and MSCs
are induced to differentiate into smooth muscle cells by placing
5.times.10.sup.4/cm.sup.2 stem cells on fibronectin coated glass
chamber slides in 2% DMEM or EBM-2 (Cambrex) supplemented with 50
ng/ml PDGF-BB for 14 days. The SMC marker, mouse anti-human
alpha-smooth muscle actin (Abcam), is used.
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