Compositions and methods for obtaining cells to treat heart tissue

Abstract

This document relates to compositions containing cardiogenic factors, to methods to obtain cells by culturing initial cells in the presence of such factors; and methods of administering the obtained cells to heart tissue.

Claims

1. A composition for use in differentiation of stem cells into cardioprogenitor cells consisting of: TGF-1; a BMP polypeptide, wherein the BMP polypeptide is selected from the group consisting of: BMP2 and BMP4; -thrombin; FGF-2; IGF-1; Activin-A; Cardiotrophin; and Cardiogenol C.

2. The composition of claim 1, wherein the BMP polypeptide is BMP4.

3. The composition of claim 1, wherein when one compound is present in said composition, it is present in an amount of between 1 and 5 ng of said TGF-1 per mL, between 1 and 10 ng of said BMP4 per mL, between 0.5 and 5 ng of said Cardiotrophin per mL, between 0.5 and 5 units of said -thrombin per mL, between 50 and 500 nM of said Cardiogenol C, between 1 and 10 ng of said FGF-2 per mL, between 10 and 100 ng of said IGF-1 per mL, and between 1 and 50 ng of said Activin-A per mL.

4. The composition of claim 1, containing 2.5 ng/mL of recombinant TGF-1, 5 ng/mL of BMP4, 1 ng/mL of Cardiotrophin, and 100 nM of Cardiogenol C.

5. The composition of claim 4, further comprising 1 U/mL of -thrombin, 10 ng/mL of FGF-2, 50 ng/mL of IGF-1, and 5 ng/mL of Activin-A.

6. The composition of claim 1, which is comprised in a medium selected from the group consisting of media containing fetal calf serum, media containing human serum, media containing platelet lysate, and media containing mixtures thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 represents the change of ejection fraction (EF) in %, considered before and after treatment with nave human mesenchymal stem cells (hMSCs) derived from bone narrow of 11 patients with coronary artery disease.

(2) FIG. 2 obtained on confocal microscopy after immunostaining with DAPI, shows protein expression of the cardiac transcription factor content for patient 2 that demonstrated no positive ejection fraction change (left) and for patient 9 (right) that demonstrated a positive ejection fraction change after treatment.

(3) FIG. 3 shows the mRNA expression of two significant cardiac transcription factors mRNA in the hMSCs of the eleven patients.

(4) FIG. 4 shows the mRNA expression, in arbitrary units (A.U.) of cardiac transcription factors for respectively Nkx2.5 mRNA, GATA-6 mRNA and Fog-1 mRNA, of untreated, nave hMSCs (left) and on CP-hMSCs, treated with a cardiogenic cocktail (right).

(5) FIG. 5 shows images obtained on confocal microscopy showing nuclear translocation of Nkx2.5, MEF2C, FOG-2 and GATA4 polypeptides in CP-hMSCs treated with a cardiogenic cocktail (right), compared with nave hMSCs (left).

(6) FIG. 6 illustrates the progressive conversion (at days D0, D5, D15, and day D20) from nave hMSCs to cocktail-guided CP-hMSCs and eventually cardiomyocytes (CM).

(7) FIG. 7 shows the transition electron microscopy ultra structure of nave hMSC and cocktail-guided cardiomyocyte.

(8) FIG. 8 shows a cocktail-guided cardiomyocyte in light microscopy.

(9) FIG. 9 represents, on the first graph on the left, the Tbx-5 mRNA expression (in AU) for nave hMSC and CP-hMSC from each patient of FIG. 1, the results for the nave cells being on the right and the one for the CP cells being on the left of the histogram. The second graph in the middle presents MEF2C mRNA expression, and the third graph on the right presents MESP-1 mRNA expression. The average values for the nave cells from all patients and the one for the CP cells for all patients are given in the background of the histogram.

(10) FIG. 10 is a graph which represents the ejection fraction change (AEjection Fraction) after treatment of heart with different quantities of nave hMSC (right part of histogram for each of twelve patients) versus treatment of hearts with CP-hMSCs, i.e. cocktail-guided hMSC (CP-hMSC).

(11) FIG. 11 represents the echocardiography of infarcted hearts untreated (left) and treated with cardiopoietic cells (right), which shows a far better anterior wall reanimation upon treatment with CP-hMSCs.

(12) FIG. 12 is a graph similar to FIG. 5 showing the ejection fraction change (EF) after injection of nave (on the left) and cardiopoietic cells (on the right) into infracted myocardium. Sham is injection without cells.

(13) FIG. 13 shows murine infarcted hearts treated with nave hMSC and CP-hMSCs six months following treatment initiation. Aneurysms and scar, which remained uncorrected in nave hMSCs treated hearts, were resolved with CP-hMSCs treatment that induced re-muscularization.

(14) FIG. 14 shows that confocal resolution revealed, in the cardiopoietic hMSC treated murine myocardium, widespread presence of human-derived cells with positive staining for h-ALU-DNA sequences specific to the human species validated with human-specific lamin immunostaining, all of which were absent in infarcted controls.

(15) FIG. 15 shows that cardiomyocytes of human origin were tracked by co-localization of human cardiac troponin-I and -actinin in cardiopoietic hMSC-treated hearts. Cardiomyocytes of human origin were absent from nave hMSC-treated hearts. Quantification within infarcted anterior walls revealed 32% and 255% of myocardial nuclei in nave versus CP-hMSC-treated hearts, implying enhanced engraftment with cardiopoietic hMSC treatment.

(16) FIG. 16 contains photographs of nave and CP hMSC stained for human troponin, ventricular myosin light chain mIC2V, and DAPI. Ventricular cell phenotype was corroborated with counter staining of human-troponin positive cells with ventricular myosin light chain mIC2V immunostaining in repaired anterior wall, as shown in FIG. 15 or resolving scar as shown in FIG. 17.

(17) FIG. 17 is a photograph of a remnant scar stained for human troponin, ventricular myosin light chain mIC2V, and DAPI.

(18) FIG. 18 contains photographs of CP-hMSCs-treated regenerating myocardium demonstrating angiogenesis distal to the occluded coronary vessel.

(19) FIG. 19 demonstrates CP-hMSCs contribution to neo-vascularization via expression of human PECAM-1 (CD-31) within the myocardial vasculature.

(20) FIG. 20 is a graph plotting ejection fraction relative to sham (%) versus time (months) following cell delivery. The long-term impact of CP-hMSCs treatment was tracked for more than one 1-year, or one third of murine lifespan which would translate into 25-years of human life. Relative to sham, treatment with nave hMSC showed a 5% and 2.5% ejection fraction effect at 6 and 12 months, respectively. In contrast, cardiopoietic hMSC treated infarcted mice demonstrated significant ejection fraction improvement of 25% at 6 and 12 months relative to sham.

(21) FIG. 21 is a graph plotting ejection fraction (%) versus time (months) post cell transplantation. The infarcted cohort was stratified to evaluate efficacy in subgroups with documented overt heart failure (ejection fraction <45%) at the time of intervention. Despite equivalent pre-treatment ejection fraction at 35%, only cardiopoietic hMSC treatment improved absolute ejection fraction by 10% at 6 and 12-months, in contrast to a 5% decline in ejection fraction in the nave hMSC-treated cohort.

(22) FIG. 22 is a bar graph plotting survivorship (%) for the indicated subgroups of mice. In overt heart failure subgroups at 400 days follow-up, no survivors were present in the sham and mortality of >50% was recorded with nave hMSC treatment. In contrast, a >80% survival was attained with cardiopoietic hMSC treatment.

(23) FIG. 23 illustrates the safety of treatment with CP-hMSCs, determined by pathological examination and electrocardiography.

EXAMPLES

(24) Patients undergoing coronary artery bypass for ischemic heart disease were randomly selected for bone marrow harvest. They provided informed consent, and study protocols were approved by pertinent Institutional Ethics Committee and Institutional Animal Care and Use Committee. Is worth noting that no injections were made to patients but to mice.

Example 1

(25) Mesenchymal stem cells were derived from human bone marrow withdrawn from the posterior iliac crest of the pelvic bone of 18- to 45-year-old healthy individuals (Cambrex, East Rutherford, N.J.). Based on flow cytometry analysis, the mesenchymal stem cells expressed CD90, CD133, CD105, CD166, CD29, and CD44, and did not express CD14, CD34, and CD45.

(26) Human bone marrow-derived mesenchymal stem cells were cultured in either platelet lysate or serum supplemented with TGF-1 (2.5 ng/ml), BMP4 (5 ng/ml), FGF-2 (5 ng/ml), IGF-1 (50 ng/ml), Activin-A (10 ng/ml), Cardiotrophin (1 ng/ml), -thrombin (1 Unit/ml), and Cardiogenol C (100 nM). After 4-10 days in the platelet lysate-containing culture at a density of about 1000-2000 cells per cm.sup.2, the cells were found to express 2-5-fold more MEF2c mRNA, MESP-1 mRNA, Tbx-5 mRNA, GATA6 mRNA, Flk-1 or FOG 1 mRNA than untreated mesenchymal stem cells.

(27) After 5-15 days in the serum-containing culture at a density of about 1000-2000 cells per cm.sup.2, the cells were found to express 5-10-fold more MEF2c mRNA, MESP-1 mRNA, Tbx-5 mRNA, GATA 4 mRNA, GATA6 mRNA, Flk-1 or FOG 1 mRNA than untreated mesenchymal stem cells.

(28) The primer pairs used for the RT-PCR analysis were standard primers obtained commercially from Applied Biosystems.

(29) Results demonstrating that the differentiated cardioprogenitor cells have the ability to incorporate into heart tissue as functional cardiomyocytes were obtained both in vivo within the beating heart, and in vitro following autopsy. In vivo, under isoflurane anesthesia, direct myocardial delivery of cardioprogenitor cells into diseased hearts improved cardiac performance as monitored by echocardiography in the short axis with a two-dimensional M-mode probing in the long axis, Doppler pulse wave analysis, and 12-lead electrocardiography.

(30) Harvested heart tissue was fixed in 3% paraformaldehyde, sectioned, and subjected to immuno-probing for human cell tracking. New human derived cardiomyocytes and vasculature, with functional improvement and scar resolution, was documented on analysis in mice treated with cardioprogenitor cells fulfilling release criteria (e.g., elevated level of expression of MEF2c mRNA, MESP-1 mRNA, Tbx-5 mRNA, GATA 4 mRNA, GATA6 mRNA, Flk-1 or FOG 1 mRNA), in contrast to absence of benefit with cells that did not pass the release criteria.

(31) In order to scale-up the production of cardiopoietic cells for autologous injection in patients, an alternative method was considered as immunofluorescence can be time-consuming, qualitative and potentially operator-dependent. One method of choice is real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). This method gives faster results (within one day) that are operator-independent and quantified relative to a reference standard. In addition, while immunostained samples require one by one fluorescent microscopy evaluation, up to 48 different samples (or conditions) can be tested in duplicate by RT-qPCR using 96-well plates.

(32) In order to identify suitable markers for RT-QPCR, cardiopoietic cells were derived from bone marrow samples obtained from cardiac patients (n=7). Cells were evaluated by immunofluorescence staining for MEF2C and Nkx2.5. RNA was extracted from these cells and expression of Nkx2.5 and MEF2C measured by real-time quantitative PCR.

(33) The reference standard consisted of cells from the same batch not cultured in the presence of the cardiogenic cocktail.

(34) Results were calculated using the double delta-Ct method normalizing the data obtained from treated cells to those from untreated cells.

(35) MEF2C was identified as suitable marker of cardiopoietic cells by both qPCR and immunofluorescence (nuclear translocation) when compared to naive cells. By contrast, the qualitative change in Nkx2.5 seen at the protein level by immunofluorescence (nuclear translocation) was initially not translated into a quantitative change at the RNA level relative to untreated cells. Genes downstream of Nkx2.5 were then investigated, since induction of their expression would depend on nuclear translocation of Nxk2.5. This led to the identification of MESP-1, Flk-1 and Tbx5 as additional suitable genes for identification by QPCR.

(36) Human bone marrow aspirates (15-20 ml) were obtained during coronary artery bypass surgery following sternotomy. Bone marrow was cryostored in a DMSO-based serum-free freezing solution. Mesenchymal stem cells were recruited by platting of raw bone marrow on plastic dishes with a wash at 12 h selecting adhesive cells with identity confirmed by Fluorescence-Activated Cell Sorting (FACS) analysis using the CD34.sup./CD45.sup./CD133.sup.+ marker panel. Cells were cultured at 37 C. in DMEM supplemented with 5% human platelet lysate (Mayo Clinic Blood Bank, Rochester, Minn.).

(37) Myocardial infarction was performed in nude, immunocompromised mice (Harlan, Indianapolis, Ind.). Following a blinded design, one month post-infarction a total of 600,000 nave or cardiopoiesis guided hMSC, suspended in 12.5 l of propagation medium, was injected under microscopic visualization in five epicardial sites on the anterior wall of the left ventricle. Sham underwent the same surgical procedure without cell injection. Myocardial injection of bone marrow hMSC into this chronic infarction model demonstrated heterogeneity in outcome with transplantation of cells from only two out of the eleven studied individuals improving ejection fraction on echocardiography.

(38) Patients 3 and 9 were identified as individuals with a high cardio-generative potential. It was first observed from FIG. 1 that the change of ejection fraction in mice (n=3) treated with hMSC from each patients 3 and 9 was significantly positive, whereas the change for each other patient was not.

(39) The protein expression of cardiac transcription factors was observed in hMSC on confocal microscopy, as shown in FIG. 2. Bar corresponds to 20 m representative for all panels.

(40) Immunostaining was performed with antibodies specific for MEF2C (1:400, Cell Signaling Technologies, Danvers, Mass.), Nkx2.5 (1:150, Santa Cruz Biotechnology Inc., Santa Cruz, Calif.), GATA4 (Santa Cruz Biotechnology Inc.), Phospho-AKT.sup.Ser473 (1:100, Cell Signaling Technologies), Tbx5 (1:5000, Abcam, Cambridge, Mass.), Mesp-1 (1:250, Novus Bio, Littleton, Colo.), Fog-2 (1:100, Santa Cruz Biotechnology), sarcomeric protein -actinin (1:500, Sigma-Aldrich) and human-specific Troponin-I (1:100, Abcam), mIC2v (1:500, Synaptic Systems, Gottigen, Germany), Sca-1 (1:100, R&D Systems, Minneapolis, Minn.), CD-31/PECAM-1 (1:500, Beckman Coulter, Fullerton, Calif.), -smooth muscle actin (Abcam), human-specific Troponin-I (1:100, Abcam), human Lamin A/C (1:50, Novacastra, New Castle, UK), and Ki67 (1:500, Abcam) following fixation in 3% paraformaldehyde and permeabilization with 1% Triton X-100, and along with DAPI staining to visualize nuclei on confocal microscopy performed with a LSM 510 Laser scanning confocal microscope (Carl Zeiss Inc., Jena, Germany).

(41) Early cardiac transcription factors Nkx 2.5, Tbx-5 and MESP1 late cardiac transcription factor MEF2C were observed under staining with DAPI. The results for patient 2 are on the left, the one for patient 9 on the right. The images obtained show that the expression of the cardiac transcription factors is weak for the hMSC from patient 2 and high for the one of patient 9. This corroborates the fact that the hMSC from patient 9 give an efficient therapeutic benefit whereas the hMSC from patient 2 do not. The coloration afforded by DAPI is blue.

(42) On FIG. 2 the first series of images for Nkx 2.5 show the nuclei of the cells colored DAPI (left) solely blue for the hMSC of patient 2 (left). A weak green colouration corresponding to the presence of Nkx 2.5 in the cytoplasm also appears. The corresponding image for patient 9 (right) shows a higher expression of Nkx 2.5 (green) in the cytoplasm and also in the nuclei of the cells.

(43) The second series of images show the cardiac transcription factors Tbx-5 (green) and MESP-1 (red) for patient 2, the nuclei of the cells and coloured in blue by the DAPI, no green or red colour is visible, which corresponds to no expression of TbX-5 and MESP-1. For patient 2, the cytoplasms of the cells are coloured in red and the nuclei in green, which corresponds to strong expression of both cardiac transcription factors and to a translocation of Tbx-5 to the nuclei of the cells.

(44) The third series of images gives results for MEF2C similar to the one for Nkx 2.5.

(45) FIG. 3 shows the mRNA expression studied in qPCR revealing cardiac transcription factor expression (MEF2C and Tbx-5) for the hMSC of the eleven patients of the study.

(46) Quantitative polymerase chain reaction (qPCR) was performed using a TaqMan PCR kit with an Applied Biosystems 7,900HT Sequence Detection System (Applied Biosystems, Foster City, Calif.). TaqMan Gene Expression reactions were incubated in a 96-well plate and run in triplicate. The threshold cycle (C.sub.T) was defined as the fractional cycle number at which fluorescence passes a fixed threshold. TaqMan C.sub.T values were converted into relative fold changes determined using the 2.sup.C.sup.T method, normalized to GAPDH (P/N 435,2662-0506003) expression.

(47) Genes listed in Table 1, which are representative of cardiac transcriptional activity were evaluated.

(48) Cells were evaluated at the mRNA and protein levels prior to and following a 5-day stimulation with a cardiogenic cocktail comprising human recombinant TGF-1 (2.5 ng/ml), BMP4 (5 ng/ml), Cardiotrophin (1 ng/ml), -thrombin (1 U/ml), and Cardiogenol C (100 nM). Both the MEF2C mRNA and the Tbx-5 mRNA expressions (in arbitrary units AU) are much higher for the hMSC of patients 2 and 9 than for the one of other patients.

(49) TABLE-US-00001 TABLE 1 Applied Biosystems Assay ID Gene name Gene symbol Hs00231763_m1 Homeobox transcription factor or Nkx2.5 or NKX2-5 NK2 transcription factor related, locus 5 or NKX2.5 Hs00171403_m1 zinc finger cardiac transcription factor or GATA-4 or, GATA binding protein 4 GATA4 (AB) Hs00231149_m1 myocyte enhancer factor 2C MEF2c or MEF2C Hs00361155_m1 T-box transcription factor or Tbx5 or TBX5 T-box 5 Hs00542350_m1 GATA co-factor (Friend of GATA) or FOG 1 of FOG-1 zinc finger protein, multitype 1 or FOG1 Hs00251489_m1 Helix-loop-helix transcription factor Mesp1 or MESP1 mesoderm posterior 1 homolog (mouse) (AB) Hs00232018_m1 GATA binding protein 6 (AB) GATA-6 or GATA6 Hs00911699_m1 Kinase insert domain receptor (a type Flk-1, or III receptor tyrosine kinase) FLK1 or KDR

(50) Left ventricular function and structure were serially followed by transthoracic echocardiography (Sequoia 512; Siemens, Malvern, Pa. and VisualSonics Inc, Toronto, Canada). Ejection fraction (%) was calculated as [(LVVdLVVs)/LVVd]100, where LVVd is left ventricular end-diastolic volume (l), and LVVs is left ventricular end-systolic volume (l).

(51) FIG. 4 shows the mRNA expression, in arbitrary units (A.U.) of cardiac transcription factors for respectively Nkx2.5 mRNA, GATA-6 mRNA and Fog-1 mRNA, of untreated, nave hMSC (left) and on CP-hMSC, treated with a cardiogenic cocktail (right). It is clear, in each case, that the results are far better when using cells treated with a cardiogenic cocktail.

(52) FIG. 5 shows images obtained on confocal microscopy showing nuclear translocation Nkx2.5, MEF2c, GATA4 and FOG-2 polypeptides in nave CP-hMSC treated with a cardiogenic cocktail. Nkx2.5, MEF2c, GATA4 and FOG-2 appear in green and DAPI in blue. On the images of nave hMSCs, no transcription factor appears. The polypeptides are translocated on the nuclei of CP-hMSCs (right) as indicated by the concentrated green colour.

(53) FIG. 6 illustrates the progressive conversion (at days D0, D5, D15, and day D20) from nave hMSCs to cocktail-guided CP-hMSCs and eventually cardiomyocytes, CM. On D0, nuclei are coloured in blue by DAPI. On D5, MEF2C polypeptide is translocated on nuclei (green). On D15, sarcomeric -actinin is present (red), which shows that sarcomeres are present and hence that the cells are definitively engaged into the cardiomyocytic differentiation and are no longer cardiopoietic. A large quantity of troponin-1 is present in cardiomyocytes on D20 (terminal differentiation).

(54) FIG. 7 shows the transition electron microscopy ultrastructure of nave hMSC (left) and cocktail-guided cardiomyocyte (right). To this end, cells were cultured in 1% platelet lysate for 15 days The cardiomyocytes present a mitochondrial maturation, a sarcomerogenesis and formation of myotubes.

(55) FIG. 8 shows a cocktail-guided cardiomyocyte in light microscopy. Maturation of the excitation-contraction system was assessed through induction of calcium transients. To this end, cells were cultured for 15 days following 5 days of cocktail stimulation and loaded for 30 min at 37 C. with 5 M of the calcium-selective probe fluo-4-acetoxymethyl ester (Molecular Probes, Carlsbad, Calif.) for live imaging using a temperature controlled Zeiss LSM 510 microscope (Zeiss) and line-scan images acquired during 1 Hz stimulation.

(56) FIG. 9 shows a 3-, 8-, and 8-fold increase in Tbx-5, MEF2C and MESP-1 in treated versus untreated hMSC.

(57) As shown in FIG. 10, CP-hMSCs meeting CARPI criteria, were delivered in vivo one-month following infarction and significantly improved ejection fraction over nave patient-matched hMSC.

(58) FIG. 11 represents an echocardiography of infarcted hearts untreated (left) and treated with cardiopoietic cells (right), which shows a far better anterior wall reanimation upon treatment with CP-hMSCs. Electrocardiograms were recorded using four-limb-lead electrocardiography (MP150; Biopac, Goleta, Calif.) under light anaesthesia (1.5% isoflurane).

(59) On echocardiography, contractility improved by 15% and 20% at one- and two-months, respectively, following treatment with CP-hMSC (n=14), in contrast to marginal change recorded with nave hMSC (n=17) or sham (n=10; FIG. 9). On top: Echocardiography of infarcted hearts 4 weeks following coronary ligation and 1 day prior to cellular transplant (4 wks post MIno Tx) revealed on M-Mode an akinetic anterior wall in both study groups. Middle: 4 weeks after cellular transplantation (4 wks post Cell Tx), nave hMSC treated hearts demonstrated maintained akinesis in the anterior wall, in contrast to re-animation in the CP-hMSCs treated group. Lower: 8 weeks following cellular transplantation (8 wks post Cell Tx), nave hMSC treated hearts continued to show limited evidence for myocardial repair versus robust contractile activity in the CP hMSC treated infarcted hearts. Left panels represent para-sternal (PS) long axis views, with dash line indicating level of 2-D M-Mode capture. Arrowheads in right panels indicate anterior wall re-animation.

(60) FIG. 12 shows that on average, guided cardiopoietic hMSC achieved a marked improvement at the one and two month follow-up following injection into infarcted myocardium. In contrast, nave hMSC or sham controls had limited impact on ejection fraction. Star and double star indicate a p<0.01 over nave hMSC for the two time points.

(61) In hearts treated with cardiopoietic cells derived from hMSC, functional improvement correlated on three-month and 18-month histopathological evaluation with myocardial regeneration. Aneurysms and scar, which remained uncorrected in nave hMSC-treated hearts, were resolved with cardiopoietic hMSC treatment that induced re-muscularization (FIG. 13).

(62) Gross pathological evaluation demonstrated resolution of scar downstream of left anterior descending (LAD) artery ligation (yellow circle on the hearts) with, on cross-section, robust re-muscularization and diminished remodeling in cardiopoietic (CP, right) in contrast to nave (left) hMSC-treated infarcted hearts at 6-months following treatment initiation. These results are particularly good.

(63) Probing for ALU-DNA was performed using human ALU-Probe (Biogenex, San Ramon, Calif.) by hybridization at 85 C. for 5-10 minutes and incubation at 37 C. overnight followed by anti-Fluorescein GFP-labeled secondary detection.

(64) Confocal resolution revealed, in the CP-hMSC-treated murine myocardium, widespread presence of human-derived cells with positive staining for ALU DNA sequences specific to the human species validated with human-specific lamin immunostaining, all absent in infarcted controls (FIG. 14).

(65) In contrast to sham (left), cardiopoietic hMSC treated hearts on confocal microscopy evaluation revealed dramatic presence of human nuclei as stained by a human h-ALU DNA probe (middle) imbedded within the murine infarcted myocardium, further confirmed with additional staining for a human-specific lamin antibody (right, inset shown in FIG. 14). Frozen myocardial sections were made from super-oxygenated 3% paraformaldehyde in PBS perfusion fixed hearts. Bar indicates 50 m.

(66) FIG. 15 shows that human-specific troponin-I antibody revealed no staining in nave (left) versus significant staining in the anterior wall of cardiopoietic hMSC treated hearts (middle and right panels)

(67) Moreover, as shown in FIG. 16 human troponin-I staining of nave (top) versus cardiopoietic (bottom) hMSC treated hearts, counterstained with mIC2v, demonstrated generation of ventricular myocardium from engrafted human cells. Bars indicate 20 m (top) and 50 m (bottom).

(68) As illustrated in FIG. 17 within the remnant scar of cardiopoietic derived from hMSC-treated hearts, human stem cell derived myocardium could be distinguished from native murine myocardium with human troponin colocalization with mIC2V. Bar indicates 50 m.

(69) In FIG. 18 surface microscopy detected angiogenesis distal to the ligated LAD (black circle) in CP-hMSCs-treated hearts arising from the right coronary artery (RCA; left bottom) and circumflex (right bottom).

(70) FIG. 19 shows confocal evaluation of collateral vessels from cardiopoietic hMSC treated hearts demonstrated human-specific CD-31 (PECAM-1) staining. Bar represents 20 m.

(71) FIG. 20 shows the evolution of the change of ejection fraction relative to sham in %, during 12 months, for treatment with both nave and cocktail-guided (CP) hMSC. Relative to sham, treatment with nave hMSC showed a 5% and 2.5% ejection fraction effect at 6 and 12 months, respectively.

(72) In contrast, CP-hMSCs-treated infarcted mice demonstrated significant ejection fraction improvement of 25% at 6 and 12 months relative to sham (FIG. 20). Furthermore, the infarcted cohort was stratified to evaluate efficacy in subgroups with documented overt heart failure (ejection fraction <45%) at the time of intervention. Despite equivalent pre-treatment ejection fraction at 35%, only cardiopoietic hMSC treatment improved absolute ejection fraction by 10% at 6 and 12-months, in contrast to a 5% decline in ejection fraction in the nave hMSC-treated cohort (FIG. 23). As shown in FIG. 22, superior survival benefit in the cardiopoietic hMSC treated group in contrast to nave treated cohort and sham was determined through application of the Kaplan-Meier function with censoring.

(73) Efficacy of cardiopoietic (CP) hMSC was demonstrated by echocardiography at 1-year follow-up (see FIG. 25). Long axis imaging of nave stem cell treated hearts revealed a fibrotic and hypokinetic anterior wall most evident on apical M-Mode evaluation (Patient 11, left panels). In contrast, CP-hMSC-treated hearts revealed a robust contractile profile throughout the anterior wall reflecting a sustained benefit from guided stem cell therapy (Patient 11, right panels).

Example 2

(74) Similar results have been observed by treating stem cells with a cocktail containing recombinant TGF-1 (2.5 ng/ml), BMP4 (5 ng/ml), Cardiotrophin (1 ng/ml), Cardiogenol C (100 nM) and -thrombin, (1 U/ml), FGF-2 (10 ng/ml), IGF-1 (50 ng/ml) and Activin-A (5 ng/ml) used in a combinatorial fashion.

Example 3

(75) Similar results have been observed by treating stem cells with a cocktail containing recombinant TGF-1 (2.5 ng/ml), BMP-4 (5 ng/ml), Activin-A (5 ng/ml), FGF-2 (10 ng/ml), IL-6 (100 ng/ml), Factor IIa (h-thrombin, 1 U/ml), IGF-1 (50 ng/ml), and retinoic acid (1 M) used in a combinatorial fashion.

Other Embodiments

(76) It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.