U.S. patent application number 13/245787 was filed with the patent office on 2012-01-26 for cardiac stem cell populations for repair of cardiac tissue.
This patent application is currently assigned to UNIVERSITA DEGLI STUDI DI ROMA "LA SAPIENZA". Invention is credited to Massimo Battaglia, Giacomo Frati, Alessandro Giacomello, Elisa Messina.
Application Number | 20120021019 13/245787 |
Document ID | / |
Family ID | 29765944 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120021019 |
Kind Code |
A1 |
Giacomello; Alessandro ; et
al. |
January 26, 2012 |
CARDIAC STEM CELL POPULATIONS FOR REPAIR OF CARDIAC TISSUE
Abstract
Method for the isolation, expansion and preservation of cardiac
stem cells from human or animal tissue biopsy samples to be
employed in cell transplantation and functional repair of the
myocardium or other organs. Cells may also be used in gene therapy
for treating cardiomyopathies, for treating ischemic heart diseases
and for setting in vitro models to study drugs.
Inventors: |
Giacomello; Alessandro;
(Rome, IT) ; Messina; Elisa; (Rome, IT) ;
Battaglia; Massimo; (Rome, IT) ; Frati; Giacomo;
(Rome, IT) |
Assignee: |
UNIVERSITA DEGLI STUDI DI ROMA "LA
SAPIENZA"
Rome
IT
|
Family ID: |
29765944 |
Appl. No.: |
13/245787 |
Filed: |
September 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10567008 |
Jul 13, 2006 |
|
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PCT/IT04/00421 |
Jul 29, 2004 |
|
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13245787 |
|
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Current U.S.
Class: |
424/400 ;
424/93.7 |
Current CPC
Class: |
C12N 5/0657 20130101;
C12N 5/069 20130101; A61K 35/12 20130101; C12N 2501/11 20130101;
C12N 2501/175 20130101; A61P 9/10 20180101; A61P 9/00 20180101;
C12N 2501/115 20130101; C12N 2533/32 20130101 |
Class at
Publication: |
424/400 ;
424/93.7 |
International
Class: |
A61K 9/16 20060101
A61K009/16; A61P 9/00 20060101 A61P009/00; A61P 9/10 20060101
A61P009/10; A61K 35/34 20060101 A61K035/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2003 |
IT |
RM2003A000376 |
Claims
1. A population of cardiospheres for administration to a subject
for the repair of damaged cardiac tissue, comprising: cardiospheres
isolated from said subject and expanded in culture, wherein the
cardiospheres are spherical multicellular aggregates about 20
microns to about 150 microns in size, wherein the cardiospheres
comprise a mixed population of cells that generates only tissue
present in the heart, thereby reducing the risk of tumor formation,
wherein the mixed population of cells generates cardiomyocytes,
endothelial cells and smooth muscle and is therefore able to repair
both damaged muscle and vessels of the heart, wherein the
cardiospheres express a stem cell marker, wherein the cardiospheres
express at least one endothelial marker selected from the group
consisting of KDR, flk-1, and CD31.
2. The cardiospheres of claim 1, wherein the cardiospheres further
express one or more of cardiac troponin, cardiac myosin heavy
chain, and von Willebrand factor.
3. The cardiospheres of claim 1, wherein said cardiospheres express
said stem cell marker within 12 hours of said cardiospheres being
generated.
4. The cardiospheres of claim 3, said stem cell marker is CD34.
5. The cardiospheres of claim 1, wherein said cardiospheres express
at least one of said KDR, flk-1 and CD31 within 12 hours of said
cardiospheres being generated.
6. The cardiospheres of claim 1, wherein said cardiospheres are
clonally derived.
7. The cardiospheres of claim 1, wherein said cardiospheres are
dissociated into a plurality of single cells prior to
administration.
8. The cardiospheres of claim 7, wherein said cardiospheres or
single cells are able to generate continuous cell lines following
spontaneous transformation or transformation induced by chemical,
physical or biologic agents.
9. The cardiospheres of claim 7, wherein said cardiospheres or the
single cells are fused with other cells prior to
administration.
10. The cardiospheres of claim 7, wherein said cardiospheres or the
single cells are used as a source or as a recipient for nuclear
transfer to or from other cells prior to administration.
11. The cardiospheres of claim 1, wherein said cardiospheres are
expanded on a solid support.
12. The cardiospheres of claim 11, wherein said solid support
comprises a biodegradable support or a biocompatible support.
13. The cardiospheres of claim 11, wherein said solid support
comprises a surface treated with one or more of collagen and
matrigel.
14. The cardiospheres of claim 1, wherein no reprogramming of said
cardiospheres is required to achieve said repair of damaged cardiac
tissue.
15. The cardiospheres of claim 1, wherein said expansion allows
replacement of a greater amount of cardiac tissue than the amount
of cardiac tissue from which the cardiospheres were derived.
16. The cardiospheres of claim 1, wherein said cardiospheres retain
their functional properties after cryopreservation.
17. A population of cardiospheres for the repair of damaged cardiac
tissue, comprising: cardiospheres isolated from non-embryonic
cardiac tissue and expanded in culture, wherein the cardiospheres
are spherical multicellular aggregates, wherein the cardiospheres
are clonally derived, wherein the cardiospheres comprise a mixed
population of cells that generates cardiomyocytes, endothelial
cells and smooth muscle and is therefore able to repair both
damaged muscle and vessels of the heart, wherein the cardiospheres
express a stem cell marker, wherein the cardiospheres express at
least one endothelial marker selected from the group consisting of
MDR, flk-1, and CD31, and wherein the cardiospheres express one or
more of cardiac troponin, cardiac myosin heavy chain, and von
Willebrand factor.
18. The cardiospheres of claim 17, wherein the cardiospheres are
isolated by: fragmenting said non-embryonic cardiac tissue in vitro
to obtain tissue fragments; adhering at least some of the tissue
fragments to a solid support and culturing the tissue fragments in
a first culture media having nutrients, the tissue fragments sized
so that the nutrients in the media can diffuse to a substantial
portion of the tissue fragments; culturing the tissue fragments
until phase-bright cells form; isolating the phase-bright cells;
culturing the phase-bright cells on a treated substrate to obtain
one or more cardiospheres.
19. The cardiospheres of claim 17, wherein said non-embryonic
cardiac tissue is obtain from a site selected from the group
consisting of the ventricle, atrium, and auricola of the heart.
20. The cardiospheres of claim 17, wherein said damaged cardiac
tissue is a result of a myocardial infarction and wherein
administration of said cardiospheres reduces infarct size.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/567,008 filed Jul. 13, 2006 which is the U.S. National Phase
application under 35 U.S.C. .sctn.371 of International Application
PCT/IT2004/000421 filed Jul. 29, 2004, which claims priority to
Italian Application RM2003 A 000376, filed Jul. 31, 2003. The
entirety of each of these applications is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention concerns a method for the isolation and
expansion of cardiac stem cells derived from postnatal cardiac
tissue biopsy.
[0004] The invention deals with a method for the isolation,
expansion and preservation of cardiac stem cells from human or
animal tissue biopsy samples to be employed in cell transplantation
and functional repair of the myocardium or other organs.
[0005] The cells may also be used in gene therapy, for treating
genetic cardiomyopathies by expressing the healthy gene in cells
from biopsies of subjects with genetic defects, propagating the
cells in vitro and then transplanting them in the patient; for
treating ischemic heart diseases by inducing the release of
angiogenic growth factors by the transplanted cells; and for the
setting of an in vitro models to study drugs.
[0006] 2. Prior Art
[0007] Stem cells (SC) are able to replicate and to differentiate
in response to appropriate signals, thus enabling the formation or
regeneration of specialized tissues.
[0008] It was thought that cardiomyocytes were terminally
differentiated cells; however, emerging evidence has shown the
modest potential of these cells to proliferate in animal models and
in heart transplant patients (1-4).
[0009] The limited ability of adult cardiomyocytes to undergo
mitosis and to regenerate the myocardium after injury leads to a
permanent deficiency in the number of functioning cells, with the
development and progression of cardiac insufficiency. In the end
stage of the disease, the alternative treatment to transplantation
is the implantation of SC in the injured myocardium
(cardiomyoplasty). This method has produced promising results in
animal models and has been experimented also in humans. However,
the problem of having a source and an availability of SC remains
(5-7).
[0010] While embryonic SC (undifferentiated cells from the embryo
that can produce a wide range of specialized cells and that can be
derived from the cell mass inside blastocytes which, in humans,
form 4-5 days after fertilization of the ovum) have a marked
capability to proliferate and differentiate, their potential
immunogenicity, arrhythmogenicity, and ethical issues in
particular, have limited their use. Moreover, embryonic SC are
pluripotent, consequently their use carries a potential risk of
generating teratomas (as occurs in animal models). Hence, before
these cells can be used, they need to be differentiated in vitro in
cardiomyocytes.
[0011] There exist various types of cardiomyocytes (ventricular,
atrial, sinus node, Purkinje, with pacemaker functions, etc.).
Embryonic SC have the potential capability to generate these
cardiomyocyte phenotypes in vitro but the yield is insufficient.
Furthermore, the in vivo proliferative capability of cardiomyocytes
derived from embryonic SC appears to be limited by the growth of
multinucleate cells.
[0012] An alternative is to use adult SC (undifferentiated cells
found in differentiated tissue that are able to proliferate,
reproduce and differentiate into the specialized cell types of the
tissues whence they were isolated) preferably obtained from the
same patient, which would afford the advantage of allowing
autologous transplantation without the need for immunosuppressive
therapy. For this purpose, skeletal myoblasts (satellite cells)
have been employed; however, they differentiate into skeletal
myocytes with morphologic and functional properties differing from
those of the cardiac muscle. The inability of skeletal myoblasts to
transdifferentiate into cardiomyocytes and to couple with them
could give rise to arrhythmias or other anomalies
[0013] SC derived from bone marrow offer an attractive alternative.
Mesenchymal SC (MSC) of the bone marrow can differentiate into
cardiomyocytes in vitro (treated with DNA-demethylating agents) and
in vivo where, however, in the presence of fibrosis, they mostly
generate fibroblast-like cells. Hematopoietic SC (HSC) of the bone
marrow (so-called side population cells [SPcells]) are pluripotent
in that they can generate vascular epithelium, smooth muscle cells
and cardiomyocytes. But the functional and electrophysiologic
properties of HSC- and MSC-derived cardiomyocytes are not well
characterized, and the use of undifferentiated cells instead of
cardiomyocytes could give rise to in vivo differentiation into
fibroblasts rather than muscle cells or to the development of
tumors.
[0014] Although human cardiomyocytes have been conventionally
considered terminally differentiated cells (i.e. unable to re-enter
the cell cycle and to divide), indirect evidence accumulating over
the past two years has suggested the existence of adult SC in the
heart. These cells are ideal candidates for cardioplasty in that
they need no reprogramming, give rise only to cells present in the
heart, i.e. cardiomyocytes and vessels (endothelial cells and
smooth muscles) and may, because this is their physiologic
function, survive in transplant patients, integrate into the
surrounding tissues and carry out their functions for longer
periods without causing any damage. Patent applications WO
03/008535 and WO 03/006950 concern methods to derive cardiomyocytes
from embryonic SC. Patent applications WO 02/13760 and WO 02/09650
deal with the use of adult SC (particularly hematopoietic and/or
cardiac cells, without indicating a method to isolate them, also in
combination) to repair cardiac injury or in treating cardiovascular
diseases in general.
[0015] Patent application WO 99/49015 deals with the isolation of
pluripotent cardiac SC of the adult p53-/- mouse. In particular,
the description concerns the heart-derived pluripotent SC that
differentiate and proliferate to produce a variety of cell types,
including cardiocytes, fibroblasts, smooth muscle cells, skeletal
muscle cells, keratinocytes, osteoblasts and chondrocytes. The
cells may be employed in methods to treat patients with cardiac
tissue necrosis. The SC proliferate and differentiate to produce
cardiocytes that replace the necrotic tissue.
[0016] However, the method differs from that of the present
invention, which was based on the assumption that the cardiac
muscle cells, the striate muscles and the smooth muscle cells
derived from a common precursor, the myoblast. Furthermore, there
is no in vivo evidence from cardiomyopathic animals that supports
the applicability of the method. Lastly, the methods differ
substantially. In the method described in patent WO 99/49015, adult
p53-/- mouse hearts are fragmented, dissociated with DNAse and
collagenase. After centrifugation, the sediment myocytes are
isolated on a discontinuous gradient (Percoll) and plated on a
medium containing 5% FBS and then on a medium containing 15% FBS 20
days later. Between days 20 and 26, small (<5 .mu.m) round,
nonadherent, slow-growth, phase-bright cells with a high
nucleus-to-cytoplasm ratio form in the suspension. These cells
continue to live in the suspension for about 1.5 months in the
presence of 10% horse serum. Then the cells remain suspended also
without the addition of horse serum. The nonadherent SC do not form
colonies in methylcellulose and proliferate in the presence of
serum, SCF, aFGF, bFGF, and cFGF. In the absence of horse serum,
the nonadherent cells differentiate into differently appearing
adherent cells the authors have identified by mainly morphologic
criteria as cardiocytes, chondrocytes, fibroblasts, smooth muscle
cells, skeletal muscle myoblasts, pericytes, and other cells the
authors have called adherent SC. About one fourth to one fifth of
these cells is positive to alkaline phosphatase (osteoblasts and
endothelial cells); all cells are negative to acetylated LDL
(absence of endothelial cells) and to myosin heavy chain (MF20).
The cells undergo mitosis when stimulated by bFGF, aFGF and cFGF.
In the absence of serum, they differentiate into cells resembling a
fried egg (myocytes), After treatment with ascorbic acid/a-GP, they
differentiate into chondrocyte-like cells.
[0017] Adherent cells cloned by limiting dilution give rise to
mesenchymal cells, including osteoblasts, chondrocytes, adipocytes
and myocytes, although they cannot be clearly identified due to
often inappropriate morphologic criteria and markers. All the cells
tested negative to acetylated LDL (absence of endothelial cells).
None of the 11 isolated clones could be induced to differentiate
toward a single mesenchymal lineage.
[0018] The isolation of the cardiac-derived SC of neonate mice (1-4
days) is also described, wherein the passage of myocytes on human
fibronectin is added to eliminate the fibroblasts. However, no data
are given about the characteristics of the isolated SC.
Furthermore, the cells isolated with the previous method do not
give rise to the formation of an essential component of the heart
tissues, i.e. vessels and endothelium.
DESCRIPTION OF THE INVENTION
[0019] The method of the present invention employs heart biopsy
tissue as starting material, hence an elective material that cannot
be used in the method described in patent application WO 99/49015,
since the material was insufficient. After fragmenting the biopsy
specimen and possibly using dissociating agents (e.g. trypsin, EDTA
and collagenase), the fragments are plated and added to a medium
containing 10% FBS; 10-30 days later, fibroblast-like adherent
cells grow from the explants over which small round, phase-bright
cells migrate that tend to cluster but are either not or only
weakly adherent. The cells are isolated by washing and mild
dissociation (e.g. EDTA, trypsin-EDTA for 2-3 min). The cells are
then plated on polylysine-treated cellular substrates in an
appropriate medium unlike that used in the previous technique, in
that it is horse-serum-free and contains other growth factors;
after 2-3 days cell aggregates (cardiospheres) arise that tend to
grow as floating formations. The authors have found that the
cardiac-forming cells are postnatal SC that can be advantageously
employed for reimplantation in the myocardium.
[0020] These cells are able to multiply, while maintaining their
origin characteristics for a period (at least 60 days) that is long
enough to markedly enrich the cell population. Mechanical
disaggregation of the cardiospheres (CS) by repeated pipetting and
changing the medium every 3 days increases the number of CS (about
100-fold every 10 days) for at least the first 20 days. Given the
number of SC that can be derived from a biopsy and their ability to
multiply in vitro, it is thought that they can be used to replace a
greater amount of tissue than that removed.
[0021] Certain cells in the CS present stem-cell markers (ckit,
sca-1, CD34) that are able to differentiate toward the main
components of the myocardium (cardiomyocytes and vessels). As
evaluated by immunohistochemistry and/or RT-PCR, certain cells
spontaneously express, particularly at the border of the CS,
markers for cardiomyocyte (troponin I, ANP, myosin heavy chain) and
for endothelial cells (von Willebrand factor and Ve-cadherin). The
human CS, in a co-culture with rat myocytes, beat spontaneously.
When inoculated subcutaneously in SCID mice, the murine CS give
rise to growths containing cardiac muscle tissue and vessels within
several days.
[0022] The authors have thus demonstrated that the SC can be
derived in a reproducible manner from biopsy tissue of the atrium,
ventricle and auricola of human subjects aged from 1 month to 70
years. The CS pertaining to the invention can be cryopreserved, and
they maintain their functional characteristics after thawing.
[0023] Adult cardiac SC with similar characteristics can also be
isolated from the mouse. In particular, to better understand cell
differentiation in CS, several breeds of transgenic mice were
studied; the findings confirmed the results obtained with human
cells.
[0024] Lastly, the authors have shown in an animal model that human
CS can be used for cardioplasty. When inoculated in the infarcted
area (transthoracic cauterization or LAD ligation) of a SCID mouse,
the cells give rise to cardiac tissue that presents good
integration with the host tissue, as observed by morphology and
immunohistochemistry studies.
[0025] Hence, the isolation and expansion of CS by the method of
the invention is novel and advantageous compared with that
described in the previous technique in terms of the origin of the
sample, the methods of isolation and expansion and the morphologic
and functional characteristics of the derived cells.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The method comprises the following steps: biopsy sample
obtained under sterile conditions and transported to the
laboratory; preparation of fragments sized large enough to allow
diffusion of nutrients present in the culture medium; distribution
of fragments on culture plates and incubation under conditions
appropriate for cell survival and growth; sampling of culture
medium and cells and transfer to other culture plates under
conditions adequate for cell expansion.
[0027] An object of the invention is a method to obtain stem cells
able to repair damaged myocardiac tissue, comprising the following
steps:
[0028] a) take a biopsy specimen of cardiac tissue and keep it in
an appropriate culture medium;
[0029] b) treat the specimen under appropriate conditions with mild
mechanical and/or chemical and/or enzymatic techniques to obtain
tissue fragments sized large enough to allow the diffusion of
nutrients present in the medium;
[0030] c) leave the tissue fragments to adhere to appropriate solid
supports and maintain them in a medium containing convenient serum
and/or growth factors;
[0031] d) allow the cells to grow, changing the medium partially or
completely, until multicellular structures form that are either
weakly adherent or do not adhere to the support;
[0032] e) separate said multicellular structures from the rest of
the culture;
[0033] f) treat said multicellular structures by mild dissociation
until most of the small phase-bright spherical cells detach but
maintain their morphologic and functional characteristics;
[0034] g) plate the cells on culture substrates treated with
polylysine or other agents promoting the adhesion of the culture to
the support in a medium containing at least the minimal essential
constituents for the growth of mammalian cells;
[0035] h) possibly repeat steps d) to g) at least once;
[0036] i) select the cells that aggregate in phase-bright spheroid
formations (cardiospheres);
[0037] l) electively promote the formation of new cardiospheres by
mild dissociation thereof and new formation;
[0038] m) eventually cryopreserve the cardiospheres for use after
thawing.
[0039] Preferably stem cells are derived from non-embryonic cardiac
tissue biopsies.
[0040] In one embodiment of the invention at least one of the steps
follows treatment with oxygen concentrations different from that
normally present in the atmosphere in order to modify the biologic
characteristics of the cultures.
[0041] Experts in the field will understand that the CS derived
with the procedure of the invention may be able to generate
continuous cell lines following spontaneous transformation or
transformation induced by chemical, physical or biologic
agents.
[0042] In another embodiment the cells giving rise to and/or
constituting cardiospheres are fused with other cells.
[0043] In another embodiment the cells giving rise to and/or
constituting cardiospheres are used for nuclear transfer to and
from other cells.
[0044] In another embodiment the cells giving rise to and/or
constituting cardiospheres are grown in at least one stage on
biodegradable and/or biocompatible supports.
[0045] In another embodiment the cells giving rise to and/or
constituting cardiospheres are cultured in bioreactors and/or
fermenters.
[0046] It is another object of the invention cells giving rise to
and/or constituting cardiospheres able to repair myocardiac tissue
obtainable according to the method of previous claims. Preferably
said cells are to be used in gene therapy. Preferably said cells
are to be used for nuclear transfers to and from other cells. The
CS derived with the method of the invention can be variously used
in the repair of myocardiac tissue, for nuclear transfer from and
to other cells, in gene therapy for cardiopathies of genetic
origin.
BRIEF DESCRIPTION OF FIGURES
[0047] FIG. 1-CS proliferation. A.sub.1-1A.sub.4) Phase micrographs
of floating CSs (cultured from <24 h to >48 h) derived from a
primary culture of a human atrial bioptical sample.
1B.sub.1-1B.sub.3) Proliferation curves of human and mouse CSs
(human CSs were derived from 8 different subjects (1B.sub.1) and
from pre- and post-natal mouse hearts (1B.sub.2 and 1B.sub.3)
respectively), in the presence (1B.sub.1 and 1B.sub.2) and in
absence (1B.sub.3) of 3.5% serum. Number of spheres refers to the
mean number per well from which 90% of the spheres where withdrawn
at each time-point for further analysis. Note the different pattern
of proliferation between the human and mouse CSs and the rapid rise
of the curves, followed by an irreversible decline in the
serum-free conditions. IC.sub.1-1C.sub.4) Fluorescence analysis of
a single cell (IC.sub.2) (obtained from a dissociated
GFP-expressing CS), when plated by limiting dilution on
mitomycin-treated STO-fibroblast-coated 96-wells plates in CGM,
over the course of the generation of the GFP-labeled clone. This
clone could be passaged and expanded on poly-D-Lysine coat
(IC.sub.3). 1D.sub.1-1D.sub.2) x-Gal staining of a eGFP/MLC3F clone
(obtained as those human) after 48 hours exposure of growth
factors-free medium: in these conditions cells in the clone become
more flattened with many nuclei showing a blue color, demonstrating
that a differentiation process occurred.
[0048] FIG. 2--CS characterization. 2A.sub.1-2A.sub.10)
Fluorescence-confocal analysis of BrdU-labeled human CSs for
cardiac differentiation markers: 6 .mu.m scans (from the periphery
to the center of the sphere) and final pictures (small and large
images respectively). BrdU (green; 2A.sub.1-2A.sub.10), cTnI (red;
2A.sub.1-2A.sub.5) and ANP (red; 2A.sub.6-2A.sub.10).
2B.sub.1-2B.sub.4) Confocal analysis of human CSs after 12 h of
culture: CD-34 (2B.sub.4), CD-31 (2B.sub.3), KDR (2B.sub.3) and
c-Kit (2B.sub.1) labeling of CS-generating cells at the beginning
of sphere formation. 2C.sub.1-2C.sub.3) Fluorescence phenotype
analysis of human CSs (cryosections): cTnI (red; 2C.sub.1),
sarcomeric myosin (2C.sub.2) and vWf (green; 2C.sub.3).
2D.sub.1-2D.sub.2), Fluorescence phenotype analysis of human
partially dissociated-CSs, after four days of culture on collagen
coat in CEM: cTnI (red; 2D.sub.2) expression appears in the
cytoplasm of the human cells (migrated from the sphere) showing a
triangular shape with a row arrangement). 2E.sub.1-2E.sub.3)
Fluorescence analysis of partially dissociated eGFP-labeled human
CSs at 96 h of co-culture with rat cardiomyocytes: the same green
cells that showed a synchronous contraction with cardiocytes,
express cTnI. 2F.sub.1-2F.sub.3) Fluorescent analysis of
connexin-43 expression (red) in eGFP-labeled human CSs co-cultured
with rat cardiomyocytes (as in panel e): a punctuate red
fluorescence is present in the cell membrane of human cells.
2G.sub.1-2G.sub.6) Phase micrograph of CSs from MLC3F-nlacZ
(2G.sub.1-2G.sub.2) and cTnI-nlacZ mice (2G.sub.3-2G.sub.6):
nuclear lacZ expression mainly localized in the external layers of
both embryo (2G.sub.1 and 2G.sub.3/4) and adult CSs (2G.sub.2 and
2G.sub.5/6), after a short time from their formation (inserts;
2G.sub.4 and 2G.sub.6) and after a few days of culture (right and
central panels). Nuclei of cells (derived from partially
dissociated CSs, cultured for 5 days on collagen-coated surfaces)
are also blue stained. 2H) Florescence analysis of a spontaneously
differentiated mouse CS: as suggested from the synchronous
contraction showed in culture, cTnI (red) is expressed in the
sphere and the cells migrated; in the last, sarcomeres are also
evident. 2I.sub.1-2I.sub.6) Fluorescence and phase analysis of CSs
from GFP-cKit (2I.sub.1, 2I.sub.2, 2I.sub.4, and 2I.sub.5),
GFP-cKit/MLC3F-nLacZ (2I.sub.3) and GFP-cKit/cTnI-nlacZ (2I.sub.6)
mice. GFP-labeled cells were present a few minutes after their
seeding in culture with CGM, at the beginning of the generation of
the CSs, later in their inner mass and after their migration out
from the oldest adherent spheres (arrows) (upper lower and left and
central panels). GFP-labeled cells did not co-localize with the
blue-stained ones (arrows) in CSs from GFP-cKit/MLC3F-nLacZ and
GFP-cKit/cTnI-nlacZ mice; fluorescent cells were present also in
the CSs' growth area (arrows) (right upper and lower panels;
2I.sub.3 and 2I.sub.6, respectively). Fluorescence, phase (small)
and merged (large) images. 2J.sub.1-2J.sub.3, FACS analysis of
post-natal mouse CSs-derived cells. A time course at 0 and 6 days
was performed and the phenotype profile for CD34, cKit, Cd31 and
sea-1 expression was analyzed and showed as percentage of positive
events (2J.sub.1). Data are presented as mean.+-.SD (n=3).
*Indicates a statistically significant difference from TO.
[0049] FIG. 3--In vivo analysis. 3A.sub.1-3A.sub.5) Ectopic
transplantation in SCID mouse of CSs from MLC3F-nlacZ/BS-eGFP mouse
(3A.sub.1-3A.sub.5). Fluorescence analysis of unfixed cryosections
(3A.sub.1-3A.sub.2; 3A.sub.4) from the subcutaneous dorsal inoculum
(day 17): GFP-cells seemed to have migrated from the spheres while
clusters of vessel-like structures could be observed mainly in the
external area (insert). Staining for SMA of one of these
cryosections showed positive immunoreaction of the sphere and some
cells within the inoculum (3A.sub.5). 3B.sub.1-3B.sub.6)
Fluorescence (3B.sub.1, 3B.sub.2, and 3B.sub.4) and phase analysis
(3B.sub.4 and 3B.sub.5) of fixed and immuno-stained cryosections
from dorsal inoculum of CSs from MLC3F-nlacZ/CD-1 and
cTnI-lacZ/CD-1 mice: tubular structures were stained for sarcomeric
myosin and cTnI (middle and lower panels respectively). X-Gal
staining labeled the cells within and those migrating from a CS
(3B.sub.2). Endothelial markers (SMA and Ve-cadherin), stained the
vasculature ("black-holes") (3B.sub.1; see also 3A.sub.3).
3C.sub.1-3C.sub.8) Orthotopic transplantation on a SCID-bg mouse,
of cryopreserved human CSs into the viable myocardium bordering a
freshly produced infarct. Confocal analysis of cryosectioned left
ventricular heart after 18 days from the coronary ligature, shows
that (3C.sub.1-3C.sub.4) cardiomyocytes expressing MHC (red) in the
regenerating myocardium (particularly those indicated by the two
central arrows), stain positive also for lamin A/C (green) (a
specific human nuclear marker). In these cells MHC expression is
evident mainly in the perinuclear area. Lamin A/C-labeled cells
(red) are present in newly generated capillaries staining for
smooth a-actin (see 3C.sub.9-3C.sub.12), and PECAM
(3C.sub.5-3C.sub.8); connexin-43 (red), as in the co-culture
experiments, lines cytoplasmic membrane of some human cell (green)
in the regenerating myocardium. Table 1. Effect of human CSs
orthotopic transplantation on echocardiographic index of myocardial
performance. Data are presented as mean.+-.SD. Abbreviations:
LVIDd, left ventricular internal dimension at end diastole; AWThd,
anterior wall thickness; FS, fractional shortening; EF, ejection
fraction. *: vs CAL+CSs p<0.05, .sctn.: vs CALp<0.05
[0050] FIGS. 4A-4B. 4a) (left) RT-PCR analysis of human CS from
pediatric (PCS), adult (aCS) subjects and cardiac fragments (H)
(ANF, NKx2.S, Ve-cadherin, GAPDH), and 4b) (right) RT-PCR analysis
of murine CS (mCS) and of mouse heart fragments (H) (a-MHC, TnC,
cardiac a-actin, GAPDH).
METHODS AND MATERIALS
Tissue Samples
[0051] The human tissue came from myocardiac biopsies of adult or
other patients who underwent open heart surgery (aortocoronary
bypass, cardiac valve replacement, tetralogy of Fallot, ventricular
septum defect) or heart transplantation for advanced dilated
cardiomyopathy or post-infarction chronic congestive
cardiomyopathy. The murine tissue came from the hearts of
previously characterized homozygous MLC3F-nLacZ mice (8) homozygous
troponin-I-nLacZ (9) and EGFP/ckit (10) CD1-crossed mice. The mice
show localized nuclear expression (cardiac and skeletal) of the
trans gene for, B-galactosidase of the myosin light chain promoter,
a tissue-specific nuclear expression (exclusively cardiac) of the
trans gene for troponin-I and a cytoplasmic expression of the EGFP
trans gene of the ckit promoter (the gene in these cell
experiments), respectively. BS-EGFP mice (11), which show
generalized expression of cytoplasmic GFP, were used as base
strains. The crossed MLC3F-nLacZ/EGFP, MLC3F-nLac-Z/EGFP-ckit,
Tn-I-nLac-Z/EGFP-ckit mice were bred according to experimental
protocol. The human cardiac tissue biopsies were preserved in
serum-free IMDM (Euroclone) at 00 C and maintained at this
condition until arrival in the laboratory (within 2 h).
Processing, Isolation and Cryopreservation of Sphere-Forming
Cell
[0052] After careful dissection of the macroscopically visible
connective tissue, the samples were cut into 1-2 mm.sup.3 pieces,
washed 3 times with Ca.sup.++/Mg.sup.++-free phosphate buffered
solution (PBS, Invitrogen) and sequentially digested 3 times for S
min each at 370 C with 0.2% trypsin (Gibco) and 0.1% collagenase IV
(Sigma). The obtained cells, the bulk of which are elements of
contaminating blood, were discarded and the remaining tissue
fragments were washed with complete explant medium (CEM) [IMDM
supplemented with 10% fetal calf serum (FCS) (Hyclone), 100 mg/ml
penicillin, 100 U/ml streptomycin (Gibco), 2 mM L-glutamine
(Gibco), 0.1 mM 2-mercaptoethanol (Sigma). The tissue pieces were
then fixed to Petri dishes (Falcon) by light scraping with a
scalpel on a plastic surface. The explants with cultured at
37.degree. C. in 5% CO2 in complete IMDM. The murine cardiac
tissues were treated similarly, except for the embryonic hearts,
where enzyme digestion prior to explant digestion was omitted and
the organs were partially dissociated with a 25 gauge needle. After
a period of 1 to 3 weeks (depending on the origin of the sample,
i.e. a shorter period for the embryonic tissue and a longer one for
the adult tissue), a layer of fibroblast-like cells forms that
derive from or surround the explants. The explants are then
periodically treated (every 6-10 days, 4 times maximum) to isolate
the sphere-forming cells. To remove only the phase-bright cells,
which migrate from the explants to the outer cell layer, the medium
is removed, and the material is collected by washing it twice with
Ca.sup.++-Mg.sup.++-free PBS and once with 0.53 mM EDTA (Versene,
Gibco) for 1-2 min, followed by mild trypsinization with 0.5 g/L-0,
53 mM Trypsin-EDTA (Gibco) at room temperature for another 2-3 min
under visual microscopy control. After the cells are collected,
complete medium is added to the explants, whereas the cells
obtained by washing and enzymatic treatment are collected by
centrifugation (1200 rpm for 7 min) and resuspended in
cardiosphere-growing medium (CGM) (35% complete IMDM/65% DMEM-Ham's
F-12 mix with 2% B27 [Gibco], 0.1 mM 2-mercaptoethanol, 10 ng/ml
EGF (Prepotek EC, Ltd.), 40 ng/ml bFGF (prepotek EC, Ltd.), 4 nM
cardiotrophin-1 (RD), 40 nM thrombin (Sigma) (final
concentrations), antibiotics and L-Glu as in the complete medium.
Depending on the number of cells obtained (from 10.sup.4 to
4.times.10.sup.5 cells/explant), the cells were resuspended by
repipetting them and then plating about 2.times.10.sup.5 cells/ml
on poly-D-lysine (BD) coated multi-well plates. After 12-24 h,
several cells begin to divide and after 48 h, cell groups form that
are often surrounded by a thin membrane and that can grow as
floating spheres and adherent spheres. The growth medium is
partially changed every 2-3 days, and the spheres are mechanically
triturated using a pipette or 1 ml needles. For cryopreservation,
the spheres (washed in Ca.sup.++-Mg.sup.++-free PBS and Versene)
are resuspended in the freezing medium (complete IMDM/DMEM-Ham-F-12
50: 50, 5% B27, 10% DMSO). To calculate the growth curves, all the
spheres are counted during the first week of growth, and then 90%
of the spheres are removed at defined times (and used for RT-PCR or
immunohistochemical analysis); after adding CGM and mechanically
triturating the residual spheres, they are left to proliferate
until the next sampling, when they are recounted. BrdU labeling is
performed for 12 h on the newly generated spheres and at defined
times in the other spheres, as indicated (Roche). For clonal
analysis, the human CSs are infected with a third-generation
lentiviral vector, pRRLsin.PPT-PGK.GFP expressing green fluorescent
protein (GFP), as described elsewhere (12). After being washed
twice, the GFP-labeled CSs are dissociated into single cells by
trituration in Ca.sup.++/Mg.sup.++-free PBS, Versene, and 1.times.
trypsin-EDTA solutions in sequence, resuspended in CGM, and then
seeded at a presumed concentration of 1 cell/well in 96-well plates
coated with a feeder layer of mitomycin-C-treated STO fibroblasts
(2 .mu.g/ml), For differentiation on a substrate-coated surface,
Ca.sup.++/Mg.sup.++-free PBS-washed, centrifuged and partially
dissociated CSs are repeatedly pipetted and then seeded in a small
volume of CEM (200-300 .mu.l) on type I collagen- (Sigma) or
Matrigel- (Falcon) coated dishes and cultured for 3-6 days.
In Vivo Analysis
[0053] For heterotopic transplantation, about 60 pooled CS obtained
from pre- and postnatal EGFP/MLC3F-nLacZ or EGFP/TnI-nLacZ or
MLC3F/nLacZ, TnI-nLacZ mice were washed twice in PBS and suspended
in 100 .mu.l of Matrigel (BD) and subcutaneously injected into the
dorsal region of anesthetized (ketamine, 35 mg/kg i.m.) adult
NOD-SCID mice. Transplanted-cardiosphere survival and function were
monitored by direct palpation of beating through the skin. After
about 3 weeks, the mice were sacrificed and the isolated inoculum
was embedded in OCT for immunocytochemical analysis. After thawing,
10-day cultures of cryopreserved human CS derived from ventricular
and atrial cardiac explants from adult subjects were used for
orthotopic transplantation. About 20 washed and partially
dissociated CS were suspended in 3 .mu.l PBS and injected in the
infarcted myocardiac area using a 27 gauge needle and a Hamilton
syringe. Myocardiac infarction was induced as described elsewhere
(13) with slight modifications. Briefly, the recipient NOD-SCID
mice (anesthetized with ketamine [35 mg/kg]+xylazine [5 mg/kg]
i.p.) underwent transthoracic cauterization (Surgitron 140 v) with
a modified electrocautery probe inserted through the internal
intercostal muscle in the fourth intercostal space on the anterior
surface of the heart. Electrocauterization (ca. 40 W) was applied
twice for 1 sec in the cutting mode before the CS were injected
(the same volume of PBS was injected in the control mice). In some
mice myocardial infarction has been also induced by LAD ligation.
After about 3 weeks, the mice were sacrificed and the isolated
heart was embedded in OCT after extensive washing in PBS and fixing
with paraformaldehyde (4%) in PBS pH 7.4.
Immunocytochemistry
[0054] Immunocytochemistry on tissue sections and on cell cultures
was performed as described elsewhere (14) using the following
antibodies: monoclonal anti-human-cTnI, anti-human-cardiac-MHC,
anti-human nucleus and polyclonal (PAb) anti-human ANP (Chemicon);
mAb anti-CD-31, CD-34 (BD Biosciences), mAb anti-human Cripto-1
(RD), monoclonal anti-Ve-cadherin, anti-sea-I, mAb anti-mouse-cKit
(Pharmigen), mAb anti-human-c-Kit (DAKO); pAb
anti-human-von-Willebrand-factor and mAb anti-human-KDR (Sigma);
mAb MF20 and pAb anti-mouse/human MHC (14), anti-desmine and
anti-Smooth-Muscle-Actin (Sigma), mAb anti-humanimouse-cTnI (15),
donated by S. Schiaffino (Dept. of Pathology, Univ. of Padua), pAb
anti-mouse-flk-1 (Santa Cruz, USA). .beta.-galactosidase activity
was detected by light microscopy, as described elsewhere (14).
Reverse-PCR Transcription Analysis
[0055] Reverse-PCR transcription analysis was performed as
described elsewhere (16). The oligonucleotides for amplifying the
genes of the CS derived from the pediatric (PCS), adult subjects
(aCS) and heart fragments (H) were the following:
TABLE-US-00001 hNkx2,5 (150 bp) forw 5'-CTCCCAACATGACCCTGAGT-3' and
rev 5'-GAGCTCAGTCCCAGTTCCAA-3', hANF (350 bp) forw
5'-AATCAAGTTCAGAGGATGGG-3' and rev 5'-AATGCATGGGGTGGGAGAGG-3',
hVe-Cad (330 bp) forw 5'-TCTCTGTCCTCTGCACAA-3' and rev
5'-ATGCAGAGGCTCATGATG-3', hGAPDH forw 5'-GAAGAGCCAAGGACAGGTAC-3'
and rev 5'-CTGCACCACCAACTGCTTAG-3;
[0056] The oligonucleotides for amplifying the genes of the murine
CS and the heart fragments (H) were the following:
TABLE-US-00002 mMHC (302 bp) forw 5'-GAAGAGTGAGCGGCGCATCAAGGA-3'
and rev 5'-TCTGCTGGAGAGGTTATTCCTCG-3', m cardiac actin (494 bp)
forw 5'-TGTTACGTCGCCTTGGATTTTGAG-3' and rev
5'-AAGAGAGAGACATATCAGAAGC-3', m cardiac TnC (410 bp) forw
5'-AATGGATGACATCTACAAAG-3' and rev 5'-TGAGCTCTTCAATGTCATCT-3'.
mGAPDH forw 5'-CCTCTGGAAAGCTGTGGCGT-3' and rev
5'-TTGGAGGCCATGTAGGCCAT-3'
Results
Isolation and Expansion of CS
[0057] Sphere-generating cells were obtained by mild enzymatic
digestion of explanted human atrial or ventricular biopsies and
fetal, embryo and postnatal mouse hearts. Soon after the generation
of a layer of fibroblast-like cells from well adherent explants,
small, round phase-bright cells began to migrate over this coat.
These cells could be harvested periodically by treatment with EDTA
and mild trypsinization, and allowed to grow on
poly-D-lysine-coated culture surfaces, in a low-serum (3.5% FCS)
medium supplemented with a serum substitute (B27), growth factors
(EGF and bFGF), cardiothrophin-1 (CT-1) (17) and thrombin (18),
which, in the first week of culture, led to a seven-fold increase
in the number of spheres with respect to that obtained using the
medium supplemented with the others factors either alone or in
combination. Time course observations of cells derived from both
human and murine explants showed that, early after their seeding
(30 min), some of these cells began to divide while still in
suspension; most cells became loosely adherent, others remained in
suspension and some contaminating fibroblast-like cells attached
firmly to the poly-D-lysine coat. Cellular divisions were evident
also from the loosely adherent cell population and produced
clusters of small, round phase-bright cells [that we termed
cardiospheres (CSs)] after 10-12 hours (FIG. 1a). Within 24-36
hours from their appearance, CSs increased in size and some of them
detached from the culture surface; after 48-72 hours most CSs were
between 20 and 150 urn in size and, when not subjected to
mechanical dissociation, the largest contained dark zones within
their inner mass (FIG. 1a).
[0058] Murine CSs started a spontaneous rhythmic contractile
activity soon after their generation and maintained this function
during their life span, while human CSs did so only when
co-cultured with rat cardiomyocytes. To be sure that contraction
was a new trait acquired by the cs cells, gfp-labeled human CSs
(partially or totally dissociated) were co-cultured with
cardiomyocytes pre-stained or not with dil. Contracting gfp-labeled
cells were observed after 48 hours of co-culture; furthermore, from
this time onwards, a red color stained also the green fluorescent
cells, suggesting that a connection is created between the human
CSs and the rat cardiac cells. In fact, labeling of human
cs/rat-cardiomyocyte co-cultures (in which only human cells were
pre-labeled with gfp by lentiviral infection) with cx-43, the major
ventricular gap junction protein, demonstrated the typical
punctuate fluorescence pattern along the cytoplasmic membrane (FIG.
2f), indicating that a functional connection is created between the
two cellular populations.
[0059] CSs were found to be composed of clonally derived cells and
did not simply represent cellular aggregates. In fact, when human
CSs [expressing the green fluorescent protein (GFP) after infection
with lentiviral vectors expressing the reporter gene] or murine CSs
(derived from eGFPIMLC3F or eGFP/cTnI-mice) were dissociated and
plated as single cells on mitomycin-treated STO-fibroblast-coated
96-wells or at clonal dilution on 10 cm O Petri dishes, fluorescent
spheres that could be sub-cloned on poly-D-lysine-coated surfaces
(FIG. 1c) were generated with a 1 to 10% efficiency. These
sub-clonally derived CSs show the same functional and phenotypic
behavior in culture: after 3 days from their appearance, some
murine clones started to, and after 48 hours of culture with CEM,
the majority (6/7) of these showed expression of the lac-Z trans
gene within nuclei after specific histochemical staining (FIG. 1d),
Equally, human clones, derived from a single GFP-labeled cell,
start a synchronous beating and express cTnI after 48 hours of
co-culture with rat cardiomyocytes.
[0060] Furthermore, when BrdU was added to the culture medium,
virtually all cells in the small, and those of the inner part of
the largest CSs, were labeled (FIG. 2a), indicating that these
cells were newly generated.
[0061] Human CS-generating cells were capable of self-renewal. With
periodical dissociation, together with partial substitution of the
growth medium every 2-3 days, a log-phase expansion of spheres was
obtained (FIG. 1b). Growth was slower for mouse CSs (owing,
probably, to the more differentiated features assumed in culture
such as beating) and, serum-dependent (FIG. 1b) as for the human
ones.
[0062] As shown in FIG. 2a, confocal immunofluorescence analysis of
BrdU labeled human CSS with anti-BrdU (green) and cardiac-troponin
i (ctni) or atrial natriuretic peptide (anp) (red), revealed
BrdU-positive cells particularly in the inner part of the spheres,
while ctni- or anp-positive cells were mainly localized in the
external layers. Furthermore, several cs-cells expressed cardiac
differentiation markers (ctni, anp) while still dividing, as
indicated by BrdU incorporation (FIG. 2a), suggesting that early
cardiac differentiation already occurred during the log-phase
growth; generally, within 2-3 weeks, some spheres became adherent,
showing a more flattened morphology. Some small cells eventually
migrated out from these "sun-like" spheres in the form of adherent
(differentiated) or small, round cells that could generate new
spheres. After thawing from cryopreservation, CSs proliferated
again, maintaining their propensity to beat.
[0063] Phenotypic analysis of newly developing human and mouse CSs
revealed expression of endothelial (KR (human)/flk-1 (mouse),
CD-31) and stem-cell (CD-34, ckit, sca-1) markers. As shown in FIG.
2b, CSs at the 2-10 cell stage, strongly reacted with antibodies
against these antigens. In larger spheres, the expression pattern
of some of these markers (particularly cKit) was similar to the
BrdU labeling (positive staining in the center and in some
peripheral zones generating satellite spheres).
[0064] A time course (0 and 6 days) of the quantitative
characterization of CSs cells with these stem and endothelial
markers was performed by FACS analysis (FIG. 21: as shown, at the
beginning of their formation (T 0) the phenotype of these cells
seems to reflect the epi-fluorescent microscopy analysis with about
10% of positive staining for all four phenotypes. However, at 6
days (T6) cKit appears as the only conserved marker, suggesting
that the cKit.sup.+ cells could be the main ones contributing to
the maintenance of proliferation, while the initial positivity to
the others may reflect an early activation state, as has been
indicated for CD-34 in several system (19). Fluorescence microscopy
analysis, performed on cryo-sectioned human CSs revealed expression
of cardiac-differentiation markers (cTnI, MHC) and also of
endothelial markers [von Willebrand factor (vWf)] (FIG. 2c). When
totally or partially dissociated into single-cells and cultured on
collagen-coated dishes, in the same medium as the explants, mouse
and humans CS-derived cells assumed a typical cardiomyocyte
morphology, phenotype (FIG. d-d.sub.1, h) and function documented
(in the mouse only) by spontaneous contraction.
[0065] As aforementioned, human CSs did not beat spontaneously;
however, these began to beat within 24 h when co-cultured with
postnatal rat cardiomyocytes, losing, after this time, their
spherical shape and assuming a "sun-like" appearance. Markers of
cardiac differentiation were co-expressed within GFP in human
labeled CSs-cells (FIG. 2e).
[0066] To follow the differentiation process of CSs during-the pre-
and post-natal age, MLC3F-nlacZ and cTnI-nlacZ mice were utilized
(8,9). These mice express a form of lacZ transgene that localizes
within the nucleus under the skeletal and cardiac muscle myosin
light chain or cardiac troponin I promoter, respectively. CSs
obtained from embryonic day 9-12, fetal day 17-18, neonatal and
adult mice, showed spontaneous expression of the reporter gene in a
variable percentage (10-60%) of spheres in the different culture
conditions employed (FIG. 2e); moreover, as for the human ones,
CS-generating cells from mice expressed stern (CD-34, sea-L, cKit)
and endothelial cell markers (flk-1 CD-31).
[0067] On this basis, we utilized transgenic mice expressing the
green fluorescent protein (GFP) under the control of the c-kit
promoter (10), in order to further clarify the cellular origin of
these spheres and to follow the pattern of their growth process. As
shown in FIG. 2i, GFP-positive cells were present from the
beginning of the formation of the CSs and, albeit with reduced
fluorescence intensity, also later, within the mass of cells of the
CSs and in cells migrating from old "sun-like" adherent CSs.
Moreover, as suggested by the growth pattern of human CSs, when
satellite secondary CSs appeared to detach from the primary ones,
GFP-positive cells localized on the margins of the latter and in
the inner part of the former.
[0068] We studied this process in double-heterozygous mice obtained
from GFP-cKitIMLC3F-nlacZ or GFP-cKit/cTnI-nLacZ crossings: as
shown in FIG. 2i, beta-Gal-positivity did not co-localize with GFP
in cells present within the growing areas.
[0069] In conclusion, CSs appear to be a mixture of cardiac stern
cells, differentiating progenitors and even spontaneously
differentiated cardiomyocytes. Vascular cells were also present,
depending on the sphere's size and time in culture. It is possible
that, as for neurospheres (20), differentiating/differentiated
cells stop dividing and/or die while stern cells continue to
proliferate in an apparent asymmetric way, giving rise to many
secondary spheres and to exponential growth in vitro. Mechanical
dissociation favors this process. Death, differentiation and
responsiveness to growth factors of the different cells within the
CS, could depend on its three-dimensional architecture and on
localization within the CS (21-22). The spontaneous formation of
spheres is a known prerogative of neural stem cells, some tumor
cell lines (LM) (22), endothelial cells (23) and fetal chicken
cardiomyocytes (24). All these models (ours included), that mimic
the true three-dimensional architecture of tissues, consist of
spheroids of aggregated cells which develop a two-compartment
system composed of a surface layer of differentiated cells and a
core of unorganized cells that first proliferate and thereafter
disappear over time (perhaps through apoptotic cell death). As well
documented in fetal chick cardiomyocytes and endothelial cell
spheroid culture, three-dimensional structure affects the
sensitivity of cells to survival and growth factors (22,23). In
particular, central spheroid cells do not differentiate and are
dependent on survival factors to prevent apoptosis, while the cells
of the surface layer seem to differentiate beyond the degree that
can be obtained in two dimensional culture and become independent
of the activity of survival factors. Furthermore, cell-cell contact
and membrane-associated factors are known to be important for the
division of neural precursor cells (25), in accordance with the
notion that stem cells will only retain their pluripotency within
an appropriate environment, as suggested by the "niche" hypothesis
(26).
[0070] To investigate the survival and morpho-functional potential
of the CSs in vivo, two sets of experiments were performed: in the
first, CS cells were injected in the dorsal subcutaneous region of
SCID mice; in the second, they were injected into the hearts of
SCID-beige mice, acutely after myocardial infarction. The intention
of ectopic transplantation experiments was to study the pattern and
the behavior of growth of CSs in a neutral milieu (i.e. one without
specific cardiac induction), in order to verify their unique
potential of generation of the main cardiac cell types and to
exclude the possibility of neoplastic transformation. For these
experiments about 60 pooled spheres/inoculum/mouse from pre- and
post-natal MLC3F-nlacZ/B5-eGFP TnI-nlacZ/B5-eGFP mice or
MLC3F-nlacZ/CD-1 and cTnI-nlacZ/CD-1 mice, were employed. During
the first 10 days, beating was appreciable through the skin over
the injection site, distant from large blood vessels. On day 17,
animals were sacrificed and the inoculum recognized as a
translucent formation, grain-like in size, wrapped in ramified
vessel-like structures. Observation of unfixed cryosections by
fluorescence microscopy revealed the presence of open spheres from
which cells appeared to have migrated; clusters of "black holes",
particularly in the periphery of the structure, were evident. The
tissue contained tubular formations, surrounded by nuclei
(Hoechst-positive), identified as cardiac sarcomeres because they
were positive for cTnI and sarcomeric myosin (FIG. 3a).
Alpha-SMA-positive structures (known to be transiently expressed
during cardiomyogenesis (27)), were present in the remainder of the
spheres and associated with the vasculature (the clusters of
"black-holes"): this exhibited well-differentiated structures with
a thin endothelium expressing Ve-cadherin (FIG. 3a) and a relative
large lumen containing erythrocytes, indicating the establishment
of successful perfusion by the host. Light microscopic observation
of the inoculum, after X-Gal staining, showed strong nuclear
expression of striated muscle-specific lacZ in the remainder of the
spheres and in some cells close to them. No multi-differentiated
structures suggesting the presence of tumor formation were
observed.
[0071] To test the acquisition of functional competence and the
cardiac regenerative potential of the CSs when challenged into an
infarcted myocardium, orthotopic transplantation experiments with
human CSs were performed. To do this, thawed (cryo-preserved) adult
human CSs, coming from three atrial (one male and two female) and
one ventricular (one female) biopsies were injected into the viable
myocardium bordering a freshly produced infarct. Each mouse
received CSs from a single passage of an explant (derived from a
single subject). Four control infarcted animals were injected with
an equal volume of PBS. After eighteen days from the intervention,
the animals were sacrificed and infarct size was determined.
Infarct size was 34.9.+-.7.1 (3.6) and 31.9.+-.6.9 (3.5) in the
CS-treated group and PBS-injected group, respectively (p=n.s.).
However, echocardiography showed better preservation of the
infarcted anterior wall thickness (0.80.+-.0.29(0.15) versus
0.60.+-.0.20(0.08) p=n.s.) and particularly of FS % (36.85.+-.16.43
(8.21) versus 17.87.+-.5.95 (2.43) p<0.05) in the CS-treated
group compared to the PBS-injected group (FIG. 3-table 1).
[0072] At the time of evaluation, bands of regenerating myocardium
were present (with different degrees of organization and thickness)
throughout most of the infarcted areas, as evaluated with
hematoxilin-eosin histochemistry and MHC immunofluorescence (FIG.
3c). In the regenerating myocardium, cells expressing lamin A/C (a
specific human nuclear marker) co-localize also with cardiomyocytes
stained positive for MHC, newly generated capillaries stained for
smooth a-actin and PECAM (FIG. 3c), and with connexin-43 expressing
cells (which, as in the co-culture experiments, defines a
connection between the human cells and the regenerating
myocardium).
[0073] Thus CSs can be considered as clones of adult stem cells,
maintaining their functional properties in vitro and in vivo also
after cryo-preservation.
[0074] While this manuscript was in preparation, two papers have
been published concerning the isolation of cardiac stem or
progenitor cells from adult mammalian heart (28, 29). Isolation of
these cells was based exclusively on the expression of a stem
cell-related surface antigen: c-kit in the first paper and Sea-1 in
the second one. In the first study (28) freshly isolated
c-kit.sup.pos Lin.sup.- cells from rat heart were found to be
self-renewing, clonogenic and multipotent, exhibiting biochemical
differentiation into the myogenic, smooth muscle cell, or
endothelial cell lineage but, differently from cells grown under
the conditions described here, failed to contract spontaneously.
When injected into an ischemic heart these cells regenerated
functional myocardium. In the second study (29), Sca-1.sup.+
cKit.sup.neg cells from mice heart were induced in vitro to
differentiate toward the cardiac myogenic lineage in response to
5'-azacytidine. When given intravenously after
ischemia/reperfusion, these cells homed to injured myocardium and
differentiated into cardiomyocytes with and without fusion to host
cells. Our data obtained on GFP-cKit transgenic mice also suggest
that the adult cardiac stem cell is cKit.sup.pos. It is possible
that CSs enclose a mixed population of cells that, as a niche,
could promote the viability of cKit progenitors and contribute to
their proliferation. The data obtained in the present paper confirm
the existence of an adult cardiac stem cell. More importantly, they
demonstrate for the first time that it is possible to isolate cells
from very small fragments of human myocardium and expand these
cells in vitro many fold (reaching numbers that would be
appropriate for in vivo transplantation in patients) without
loosing their differentiation potential, thus opening previously
unforeseen opportunities for myocardial repair.
Transgenic Mice
[0075] To follow the differentiation process of CSs during the pre-
and post-natal age, MLC3F-nlacZ and TnI-nLacZ mice were utilized.
These mice express a form of the lacZ transgene that localizes
within the nucleus under the skeletal and cardiac muscle myosin
light chain or cardiac troponin-I promoter, respectively (8, 9).
CSs derived from embryonic day 9-12, fetal day 17-18, neonatal and
adult mice, showed spontaneous expression of the reporter gene in a
variable percentage (10-60%) of spheres at the different culture
conditions employed (FIG. 4a). Moreover, the mouse CS start to beat
at the start of formation (particularly in the embryo) and continue
to beat over the course of their life span. The human CS expressed
stem (CD-34, sea-L, cKit) and endothelial cell markers (flk-1,
CD-31).
[0076] In order to further clarify the cellular origin of these
spheres and to follow the pattern of their growth process, we
utilized transgenic mice expressing the green fluorescent protein
(GFP) under the control of the c-kit promoter (10). GFP-positive
cells were present from the beginning of the formation of the CSs
and, albeit with reduced fluorescence intensity, also later.
Moreover, as suggested by the growth pattern of human CSs, when
satellite secondary CSs appeared to detach from the primary ones,
GFP-positive cells localized on the margins of the latter and in
the inner part of the former. We studied this process in
double-heterozygous mice obtained from EGFP-cKit/MLC3F-nlacZ or
TnI-nLacZ crossings. As shown in FIG. 4b, beta-Gal-positivity did
not co-localize with EGFP in cells present within the growing
areas.
Genetic Phenotype
[0077] The RT-PCR panel created on murine or human CS RNA extracts
is shown in FIG. 5. A more typical profile of cardiac progenitors
seems to be that of the human samples (in log-growth phase)
compared with the murine samples, in which it is easier to have
proliferation and differentiation occurring together.
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Sequence CWU 1
1
16120DNAArtificial SequencehNkx Forward Primer 1ctcccaacat
gaccctgagt 20220DNAArtificial SequencehNkx Reverse Primer
2gagctcagtc ccagttccaa 20320DNAArtificial SequencehANF Forward
Primer 3aatcaagttc agaggatggg 20420DNAArtificial SequencehANF
Reverse Primer 4aatgcatggg gtgggagagg 20518DNAArtificial
SequencehVE-Cad Forward Primer 5tctctgtcct ctgcacaa
18618DNAArtificial SequencehVe-Cad Reverse Primer 6atgcagaggc
tcatgatg 18720DNAArtificial SequencehGAPDH Forward Primer
7gaagagccaa ggacaggtac 20820DNAArtificial SequencehGAPDH Reverse
Primer 8ctgcaccacc aactgcttag 20924DNAArtificial SequencemMHC
Forward Primer 9gaagagtgag cggcgcatca agga 241023DNAArtificial
SequencemMHC Reverse Primer 10tctgctggag aggttattcc tcg
231124DNAArtificial SequencemCardiac Actin Forward Primer
11tgttacgtcg ccttggattt tgag 241222DNAArtificial SequencemCardiac
Actin Reverse Primer 12aagagagaga catatcagaa gc 221320DNAArtificial
SequencemCardiac TnC Forward Primer 13aatggatgac atctacaaag
201420DNAArtificial SequencemCardiac TnC Reverse Primer
14tgagctcttc aatgtcatct 201520DNAArtificial SequencemGAPDH Forward
Primer 15cctctggaaa gctgtggcgt 201620DNAArtificial SequencemGAPDH
Reverse Primer 16ttggaggcca tgtaggccat 20
* * * * *