Cardiac stem cells and uses of same in cardiac repair   

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.

CROSS-REFERENCE TO RELATED APPLICATIONS

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. §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

1. Field of the Invention

The invention concerns a method for the isolation and expansion of cardiac stem cells derived from postnatal cardiac tissue biopsy.

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.

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.

2. Prior Art

Stem cells (SC) are able to replicate and to differentiate in response to appropriate signals, thus enabling the formation or regeneration of specialized tissues.

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).

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).

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.

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.

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

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.

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. 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.

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 μm) 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/α-GP, they differentiate into chondrocyte-like cells.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

An object of the invention is a method to obtain stem cells able to repair damaged myocardiac tissue, comprising the following steps:

a) take a biopsy specimen of cardiac tissue and keep it in an appropriate culture medium;

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;

c) leave the tissue fragments to adhere to appropriate solid supports and maintain them in a medium containing convenient serum and/or growth factors;

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;

e) separate said multicellular structures from the rest of the culture;

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;

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;

h) possibly repeat steps d) to g) at least once;

i) select the cells that aggregate in phase-bright spheroid formations (cardiospheres);

l) electively promote the formation of new cardiospheres by mild dissociation thereof and new formation;

m) eventually cryopreserve the cardiospheres for use after thawing.

Preferably stem cells are derived from non-embryonic cardiac tissue biopsies.

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.

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.

In another embodiment the cells giving rise to and/or constituting cardiospheres are fused with other cells.

In another embodiment the cells giving rise to and/or constituting cardiospheres are used for nuclear transfer to and from other cells.

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.

In another embodiment the cells giving rise to and/or constituting cardiospheres are cultured in bioreactors and/or fermenters.

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.

FIG. 1—CS proliferation. a) Phase micrograph of floating CSs (cultured from <24 h to >48 h) derived from a primary culture of a human atrial bioptical sample. b) Proliferation curves of human and mouse CSs [derived from 8 different subjects (top) and from pre- and post-natal hearts (bottom) respectively], in the presence (left) and in absence (right) 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. c) Fluorescence analysis of a single cell (upper-right) (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 (lower-left). d) 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.

FIG. 2—CS characterization. a) Fluorescence-confocal analysis of BrdU-labeled human CSs for cardiac differentiation markers: 6 μm scans (from the periphery to the center of the sphere) and final pictures (small and large images respectively). BrdU (green), cTnI and ANP (red). b) Confocal analysis of human CSs after 12 h of culture: CD-34, CD-31, KDR and c-Kit labeling of CS-generating cells at the beginning of sphere formation. c) Fluorescence phenotype analysis of human CSs (cryosections): cTnI (red), sarcomeric myosin and vWf (green). d-d1), Fluorescence phenotype analysis of human partially dissociated-CSs, after four days of culture on collagen coat in CEM: cTnI (red) expression appears in the cytoplasm of the human cells (migrated from the sphere) showing a triangular shape with a row arrangement). e) 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. f) 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. g) Phase micrograph of CSs from MLC3F-nlacZ and cTnI-nlacZ mice: nuclear lacZ expression mainly localized in the external layers of both embryo and adult CSs, after a short time from their formation (inserts) 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. h) 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. i) Fluorescence and phase analysis of CSs from GFP-cKit, GFP-cKitIMLC3F-nLacZ and GFP-cKit/cTnI-nlacZ 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-cKitIMLC3F-nLacZ and GFP-cKit/cTnI-nlacZ mice; fluorescent cells were present also in the CSs\' growth area (arrows) (right upper and lower panels). Fluorescence, phase (small) and merged (large) images. 1, 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. Data are presented as mean±SD (n=3). *Indicates a statistically significant difference from TO.

FIG. 3—In vivo analysis. a) Ectopic transplantation in SCID mouse of CSs from MLC3F-nlacZ/BS-eGFP mouse (upper left panels). Fluorescence analysis of unfixed cryosections (upper left small, upper right and down left large images) 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. b) Fluorescence (right) and phase analysis (left, merged) 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 (upper right). Endothelial markers (SMA and Ve-cadherin), stained the vasculature (“black-holes”) (small images). c) 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 (upper left panel) 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 (upper right panel), and PECAM (down left panel); connexin-43 (red) (down right high magnification panel), 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, §: vs CALp<0.05

FIG. 4—a) (left) RT-PCR analysis of human CS from pediatric (PCS), adult (aCS) subjects and cardiac fragments (H) (ANF, NKx2.S, Ve-cadherin, GAPDH), and b) (right) RT-PCR analysis of murine CS (mCS) and of mouse heart fragments (H) (α-MHC, TnC, cardiac a-actin, GAPDH).

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, β-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).

After careful dissection of the macroscopically visible connective tissue, the samples were cut into 1-2 mm3 pieces, washed 3 times with Ca++/Mg++-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° 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++—Mg++-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 104 to 4×105 cells/explant), the cells were resuspended by repipetting them and then plating about 2×105 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++—Mg++-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++/Mg++-free PBS, Versene, and 1× 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 μg/ml), For differentiation on a substrate-coated surface, Ca++/Mg++-free PBS-washed, centrifuged and partially dissociated CSs are repeatedly pipetted and then seeded in a small volume of CEM (200-300 μl) on type I collagen- (Sigma) or Matrigel- (Falcon) coated dishes and cultured for 3-6 days.

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 μl 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 μl 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 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). β-galactosidase activity was detected by light microscopy, as described elsewhere (14).

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:

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;

The oligonucleotides for amplifying the genes of the murine CS and the heart fragments (H) were the following:

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

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20130122063 - Nanotubes as mitochondrial uncouplers - A method of uncoupling mitochondria in a subject including administering nanotubes to the subject in a therapeutically effective amount, wherein the nanotubes are self-rectifying is provided. A method of decreasing reactive oxygen species and decreasing detrimental loading of Ca2+ into mitochondria is provided, including administering a pharmaceutically effective amount of ...

20130122064 - Ophthalmic depot formulations for periocular or suconjunctival administration - The present invention relates to ophthalmic depot formulations comprising an active agent, e.g. embedded in a pharmacologically acceptable biocompatible polymer or a lipid encapsulating agent, e.g. for periocular or subconjunctival administration. ...

20130122057 - Organophosphorous, multivalent metal compounds, and bioactive glass material macromolecular network compositions and methods - Cements containing certain small molecule amino acid phosphate compounds such as phosphoserine and certain multivalent metal compounds such as but not limited to calcium phosphate have been found to have improved properties and form a macromolecular network in the presence of a bioactive glass material that contain silicates, phosphates, and ...

20130122065 - Pharmaceutical composition - Provided herein are pharmaceutical compositions comprising an antagonist, an agonist, a seal coat, and a sequestering polymer, wherein the antagonist, agonist, seal coat and at least one sequestering polymer are all components of a single unit, and wherein the seal coat forms a layer physically separating the antagonist from the ...

20130122062 - Polymeric compositions containing ir-emitting/absorbing additives and shaped articles comprised thereof - Polymeric compositions containing additives having properties of emission and/or absorption of radiation in the long infrared region, and articles shaped therefrom are produced, including yarns and textile articles such as fabrics or knits; such additives include organic additives or inorganic fillers which have a capacity for absorption/emission of radiation in ...

20130122056 - Ratiometric combinatorial drug delivery - The present teachings include ratiometric combinatorial drug delivery including nanoparticles, multi-drug conjugates, pharmaceutical compositions, methods of producing such compositions and methods of using such compositions, including in the treatment of diseases and conditions using drug combinations. ...

20130122059 - Suspensions of cyclosporin a form 2 - Disclosed herein are methods of formulating cyclosporin A Form 2. ...

20130122053 - Synthetic triterpenoids and tricyclic-bis-enones for use in stimulating bone and cartilage growth - The present invention concerns methods for stimulating the growth and repair of bone and cartilage using synthetic triterpenoids and tricyclic-bis-enones. Examples of suitable triterpenoids include CDDO, CDDO-Me, CDDO-Im, and CDDO-Ethylamide. Examples of tricyclic-bis-enones include TBE-31 and TBE-34. ...

20130122060 - Using bucky paper as a therapeutic aid in medical applications - Methods, systems, and uses of bucky paper are provided in the present invention. These embodiments include covering medical implants with single or multiple layers of bucky paper, treating bucky paper with various therapeutics to be released through the bucky paper to a target site, shaping bucky paper into non-conventional configurations ...


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