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Definitions

• the present invention relates to the treatment of heart disease disorders through injection of mammalian cells.
• it relates to a method for quantitatively assessing the cardio-generative potential of mammalian cells, thereby allowing a good predictability of the success of repairing a heart in need.
• It also relates to a method for quantitatively assessing the modification of this cardio-generative potential and the cardiogenic potential of a treatment aiming at cellular differentiation, and a computer device comprising a processor, and a memory encoding one or more non-neural network programs coupled to the processor, wherein said programs cause the processor to perform a method, said method comprising calculating a CARPI.
• Cardiovascular diseases are leading cause of morbidity and mortality worldwide, despite advances in patient management. In contrast to tissues with high reparative capacity, heart tissue is vulnerable to irreparable damages. Cell-based regenerative cardiovascular medicine is now being pursued in the clinical setting to address heart disease disorders.
• Cardiopoietic cells have a unique phenotype: they are characterized by nuclear translocation of Nkx2.5 and MEF2C polypeptides, combined to the absence of detectable sarcomeric proteins. This cardiopoietic status corresponds to an intermediate cell phenotype, i.e. committed to the cardiac lineage but not yet fully differentiated. Non-detectable level of sarcomeric protein expression is a unique feature of cardiopoietic cells which distinguishes them from contractile and sarcomeric- containing cardiomyocyte-like cells derived from stem cells and described in other applications such as by Chunhui Xu (US 2005/0164382) and Lough et al (US 2002/0061837).
• Increased protein content of a transcription factor may not imply its subcellular localization, which could be either cytoplasmic or nuclear.
• Nuclear translocation of Nkx2.5 and MEF2C polypeptides is necessary for definitive cardiac lineage commitment. This is further explained in Behfar A. et al, (Derivation of a cardiopoietic population from human mesenchymal stem cells yields cardiac progeny, Nature Clinical Practice, 2006, 3:S78-S82). Although nuclear translocation may be qualitatively observed by immunocytochemistry or immunohistochemistry, techniques such as western blotting or Fluorescence Activated Cell Sorting (FACS) that look at total protein content are not suitable for quantitative assessment of the subcellular distribution of a polypeptide.
• FACS Fluorescence Activated Cell Sorting
• the present invention now provides such a predictive method for determining the cardio generative potential of mammalian cells which comprises the quantitative assessment of a CARdiac generation Potential Index (CARPI) as a function of the quantification of the expression of genes of said cells. It also addresses the quantitative assessment of the modification of the cardio generative potential of mammalian cells and the cardiogenic potential of a treatment aiming at cellular differentiation.
• CARPI CARdiac generation Potential Index
• the 'cardio-generative potential' of a cell designates the ability of this cell to succeed to generate heart cells, for instance cardiac myocytes.
• Cardiopoietic cells' are an intermediate cell phenotype, i.e. committed to the cardiac lineage but not yet fully differentiated. Cardiopoietic cells are characterized by nuclear translocation of Nkx2.5 and MEF2C, combined to the absence of detectable sarcomeric proteins (Behfar et al. 'Derivation of a cardiopoietic population from human mesenchymal stem yields progeny', Nature Clin. Pract., Cardiovasc. Med. (2006) 3:
• Cardiopoietic cells retain a proliferative capacity.
• Cardiopoietic cells can be derived from stems cells including for example, human adult mesenchymal stem cells [Terzic et al. US 2008/0019944), mouse embryonic stem cells ⁇ Behfar et al,
• a 'cocktail' or 'cardiogenic cocktail' designates a composition containing at least two cardiogenic substances.
• a 'cardiogenic treatment' is a treatment which improves the cardio-generative potential of a cell.
• Example of such treatment consists in putting said cell in contact with a cocktail.
• Examples of such cocktails comprise at least two substances selected in the group consisting of growth factors, cytokines, hormones and combinations thereof.
• Said at least two substances may be selected in the group consisting of bone morphogenetic proteins (BMP) such as BMP-1 , BMP-2, BMP-5, BMP-6; epidermal growth factor (EGF); erythropoietin (EPO); fibroblast growth factors (FGF) such as FGF-1 , FGF-4, FGF-5, FGF-12, FGF-13, FGF-15, FGF-20; granulocyte-colony stimulating factor (G-CSF); granulocyte-macrophage colony stimulating factor (GM-CSF); growth differentiation factor-9 (GDF-9); hepatocyte growth factor (HGF); insuline-like growth factor (IGF) such as IGF-2; myostatin (GDF-8); neurotrophins such as NT-3, NT-4, NT-1 and nerve growth factor (NGF); platelet-derived growth factor (PDGF) such as PDGF-beta, PDGF- AA, PDGF-BB; thrombopoietin (
• SDF-1 brain-derived neurotrophic factor (BDNF); periostin; angiotensin II; Flt3 ligand; glial-derived neurotrophic factor; heparin; insulin-like growth factor binding protein-3; insulin-like growth factor binding protein-5; interleukin-3; interleukin-8; midkine; progesterone; putrescine; stem cell factor; Wnt1 ; Wnt3a; Wnt ⁇ a; caspase-4; chemokine ligand 1 ; chemokine ligand 2; chemokine ligand 5; chemokine ligand 7; chemokine ligand 1 1 ; chemokine ligand 20; haptoglobin; lectin; cholesterol 25- hydroxylase; syntaxin-8; syntaxin-1 1 ; ceruloplasmin; complement component 1 ; complement component 3; integrin alpha 6; lysosomal acid lipase 1 ; ⁇ -2 microglobulin; ubiquitin; macro
• M-CSF angiopoietin
• PIGF extracellular matrix molecules
• MCP-1 extracellular matrix molecules
• CCL3 MIP-1 ⁇
• CCL4 MIP-1 ⁇
• CCL5 RANTES
• CCL7 MCP-3
• CCL20 MIP-3 ⁇
• CCL26 eotaxin-3
• CX3CL1 fractalkine
• CXCL5 ENA-78
• CXCL11 i-TAC
• CXCL1 CXCL1
• a 'cocktail-guided cell' or a 'cell guided towards cardiac differentiation' is a cell which has been put into contact with a cocktail.
• 'Differentiation' is the process by which a less specialized cell becomes a more specialized cell.
• 'Ejection fraction' means the fraction of blood pumped out during a heartbeat.
• ejection fraction refers specifically to that of the left ventricle (left ventricular ejection fraction or LVEF).
• the invention provides a method for determining the cardio-generative potential of mammalian cells or cardiogenic potential of a treatment which comprises the assessment of a CARdiac generation Potential Index (CARPI) as a function of the quantification of the expression of genes of said cells.
• CARPI CARdiac generation Potential Index
• the CARPI is a function of the quantification of messenger RNA (mRNA) levels of specific genes of said cells.
• mRNA messenger RNA
• At least one gene is chosen from the group consisting of Nkx2.5,
• the cells may be cardiac progenitor cells.
• They may also be somatic, germ, umbilical cord blood, cardiac progenitor, embryonic, and/or genetically modified cells.
• the cells can belong to one individual, and a CARPI can be assessed for those cells before and after exposing the cells to any cardiogenic treatment.
• a CARPI is assessed for cells of an individual or group of individuals versus another individual or group of individuals.
• the CARPI is a multivariate equation where the expression of genes at the mRNA level is quantified as variables.
• the equation is preferably chosen from the group consisting of polynomials functions, transcendental functions, and combinations thereof.
• a CARPI is measured to quantitatively assess the cardiogenic potential of a treatment.
• the CARPI may be put into correlation with a parameter of cardiac function.
• the invention also relates to a computer device comprising a processor, and a memory encoding one or more programs coupled to the processor, wherein the one or more programs cause the processor to perform a method, said method comprising calculating a CARPI.
• Fig. 1 shows in Y ordinate the CARPI, in arbitrary units (AU), calculated for both naive human MSC (hMSC) and cocktail guided-hMSC (CP-hMSC) on the basis of quantification of the expression of genes at the mRNA level and in X ordinate the change of LVEF ( ⁇ EF) in percent prior and after injection in mouse infarcted hearts.
• Black symbols represent individual data; open symbols represent averaged data (Avg).
• Bone marrow samples were harvested from patients undergoing coronary artery bypass for ischemic heart disease. Patients provided informed consent, as approved by competent Institutional Ethics Committees.
• Mesenchymal stem cells were recruited by plating of raw bone marrow on plastic dishes, with a wash at 12h, selecting adhesive cells with identity confirmed by
• FACS Fluorescence-Activated Cell Sorting
• Naive human bone marrow-derived mesenchymal stem cells were cultured in either platelet lysate or serum supplemented with a cardiogenic cocktail consisting in TGF ⁇ -1 (2.5 ng/ml), BMP4 (5 ng/ml), FGF2 (5 ng/ml), IGF-1 (50 ng/ml), Activin-A (10 ng/ml), Cardiotrophin (1 ng/ml), ⁇ -thrombin (1 U/ml), and Cardiogenol C (100 nM) in order to derive a cardiopoietic population.
• a cardiogenic cocktail consisting in TGF ⁇ -1 (2.5 ng/ml), BMP4 (5 ng/ml), FGF2 (5 ng/ml), IGF-1 (50 ng/ml), Activin-A (10 ng/ml), Cardiotrophin (1 ng/ml), ⁇ -thrombin (1 U/ml), and Cardiogenol C (100 nM) in order to derive a cardiopoi
• the present invention allows the quantitative assessment of the cardio- generative potential of said cardiopoietic population, by quantifying the expression of two or more genes at the RNA level.
• This invention obviates the problems of qualitative observations, issue of time, and operator-dependence, inherent to the observation of subcellular location of transcription factor polypeptides.
• One method of choice is realtime quantitative reverse transcription polymerase chain reaction (qPCR). This method gives faster results (within one day) that are operator-independent and quantified relative to a reference standard.
• qPCR realtime quantitative reverse transcription polymerase chain reaction
• This method gives faster results (within one day) that are operator-independent and quantified relative to a reference standard.
• immunostained samples require one-by-one fluorescent microscopy evaluation, up to 48 different samples (or conditions) can be tested in duplicate by qPCR using 96-well plates.
• mRNA was extracted from cardiopoietic cells that were evaluated by immunofluorescence staining.
• the reference standard consisted of cells from the same batch cultured in the absence of the cardiogenic cocktail.
• C T The threshold cycle
• TaqMan C ⁇ values were converted into relative fold changes determined using the 2 ⁇ C T method, normalized to a housekeeping gene expression, i.e. GAPDH (P/N 435,2662-0506003).
• Results for treated cells were normalized to results obtained for the corresponding reference standard.
• a CARPI which is a function of the quantification of the expression of two or more genes of said cells, was calculated as a linear average of the expression at the RNA level of Nkx2.5, Tbx-5, MEF2C, GATA-4, GATA-6, MESP-1 and FOG-1 using a calculation spreadsheet (Microsoft Excel 2007 ® , Microsoft Corporation). The following formula was used:
• CARP! - :% .Y RSA ls ⁇ i >
• hMSC-derived cardiopoietic cells The cardio-generative potential of hMSC-derived cardiopoietic cells was evaluated in nude, immunocompromised mice (Harlan, Indianapolis, IN). The protocol was approved by the competent Institutional Animal Care and Use Committee.
• Myocardial infarction was performed. Following a blinded design, one month post-infarction a total of 600,000 total viable naive hMSC or 600,000 total viable hMSC- derived cardiopoietic cells, suspended in 12.5 ⁇ l of platelet lysate-free propagation medium, were injected under microscopic visualization in five epicardial sites on the anterior wall of the left ventricle (2.5 ⁇ l per injection site). Left ventricular function and structure were serially followed by transthoracic echocardiography (Sequoia 512; Siemens, Malvern, PA and VisualSonics Inc, Toronto,
• LVEF left ventricular ejection fraction
• LVVs left ventricular end-diastolic volume ( ⁇ l)
• LVVs left ventricular end-systolic volume ( ⁇ l).
• a change of LVEF was calculated as the difference between LVEF measured one month after cell injection and LVEF measured prior to cell injection.
• Fig. 1 is a graph plotting the CARPI for each individual cell culture against the corresponding ⁇ EF for the mouse injected with the respective said individual cell culture.
• Naive hMSC small black diamonds typically demonstrated a low CARPI associated with no significant improvement in myocardial function (negative ⁇ EF) one month post-cell injection. It is worth noting rare batches of naive hMSCs innately possessing high CARPI value together with an innate regenerative potential. The average for all batches of naive hMSCs is shown by a large white triangle.
• hMSC- derived cardiopoietic cells small black squares typically demonstrated an elevated CARPI associated with robust increase in myocardial function (positive ⁇ EF).
• the average for all batches of hMSC-derived cardiopoietic cells is shown by a large white square. Averages are represented together with the corresponding 95% confidence interval.
• the inventors demonstrate that there is a positive correlation between an elevated CARPI of the cells to be injected and the change in ejection fraction after injection in the infarcted heart.
• the CARPI is a predictive index of cardio- generative potential.

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