WO2015086052A1

WO2015086052A1 - Culture media for in vitro differentiation of cardiomiocytes from adipose tissue-derived mesenchymal stem cells and method to select them

Info

Publication number: WO2015086052A1
Application number: PCT/EP2013/076144
Authority: WO
Inventors: Gianni Soldati; Greta Maria MINONZIO
Original assignee: Swiss Stem Cell Foundation
Priority date: 2013-12-10
Filing date: 2013-12-10
Publication date: 2015-06-18

Abstract

It is disclosed a method to assess cardiogenic differentiation in adipose tissue-derived stromal cells (ASCs) and to select growth media with enhanced capability to stimulate cardiocyte differentiation of ASCs, based on assessing the enhanced expression of a selected pool of markers consisting of NKX2-5, MEF2C, HAND2, HAND1, CAMTA and FOG genes/transcription factors. Growth media selected by this method, containing a mixture of suitable growth factors and cytokines are also disclosed, showing a high cardiogenic differentiation potency of ASCs.

Description

TITLE

Culture media for in vitro differentiation of cardiomiocytes from adipose tissue-derived mesenchymal stem cells and method to select them.

STATE OF THE ART

Cardiovascular diseases (CVD) are the leading and most common cause of mortality in many developing and industrialized countries, accounting for one third of all deaths globally [1 ]. The American Heart Association reported that cardiac failure was responsible for 38% of all deaths in recent years. Nearly half of all deaths associated with CVD are the result of heart failure due to ischemic cardiomyopathy events such as myocardial infarction (Ml), a pathology characterized by clot formation within a coronary artery, which prevents blood flow to the region of the heart distal to the site of occlusion. The prevalence of CVD is expected to increase to three fourths of all deaths by 2020 in part due to an increasing prevalence of risk factors related to coronary disease, such as obesity and sedentary lifestyle (metabolic syndrome), making CVD an even greater public health concern [1 ,2]. Both of these conditions result in cardiomyocytes death by apoptosis and/or necrosis. Dead cardiomyocytes are replaced by fibroblasts that divide and migrate into the damage area to form scar tissue, leading to the development of a thin ventricular wall that no longer contracts properly [3]. Formation of a fibroblastic scar initiates a series of events that lead to remodelling, hypertrophy and ultimately heart failure and further cell deaths.

The persistence of scar tissue following myocardial infarction suggests that the heart has little, if any, capacity to generate new cardiomyocytes. Unlike most other cell types, cardiac myocytes, the functional components of the heart muscle, are incapable of replication, or do so on a very small scale. As a result, when the heart is damaged, as in the case of a myocardial infarction, there is limited internal potential for repair and the patient is left with decreased cardiac function rarely improving over time. One of the approaches that could really replace scar tissue with living cardiomiocytes is the ex vivo generation of such cells, at least in form of precursors, and the delivery of these pre-differentiated cells to the heart. De novo cardiomyocyte production would be thus to provide an ex vivo, autologous source of donor cardiomyocytes.

Developmental biology and available data on the differentiation of embryonic and adult stem cells clearly indicate that the process of forming a heart requires many carefully orchestrated signals, some of which originate outside the pre-cardiac mesoderm and some in the developing heart. During gastrulation the heart is generated from a small number of mesodermal progenitor cells. These progenitors undergo complex and dynamic processes. They migrate, proliferate and differentiate to form a linear heart tube, the primitive form of a functional heart. During these processes, cells initially acquire generic cardiogenic phenotypes typical of cardiac progenitors and later exhibit more specific properties reflecting their final differentiation, for instance atrial and ventricular cells [4].

Cardiomyocyte-specific transcription factors are relevant tools to address the degree of differentiation achieved in our in vitro system. Members of the Nk family of transcription factors are expressed in all animals with contractile vascular cells and hence are crucial for myocardial development [5]: NKX2-5 is specifically required for left ventricular chamber development [6]. Gata is another family of transcription factors that interact with Nk factors to promote differentiation of cardiomyocytes, smooth muscle cells and endoderm, and plays an important role in cardiac development. Gata regulates myocardial protein expression and is required for fusion of the heart tubes in the ventral midline. Finally, GATA4 gene knockout studies demonstrate the essential role of this factor in cardiac development and coordination of the expression of downstream cardiac genes [7]. Also T-box genes play an important role in cardiac morphogenesis and TBX5 is required for atrial septation. Another important transcription factor that we considered is BAF60C: a subunit of the BAF complex. It is expressed in the developing heart and is required for cardiac morphogenesis and establishment of left-right asymmetry. MEF2C myocyte-specific enhancer factor 2C is also involved in cardiac morphogenesis and myogenesis and vascular development. Mice without a functional copy of the MEF2C gene die before birth and have abnormalities in the heart and vascular system. Other important genes are Mespl [8], which has been described as "the master regulator" of cardiac progenitor specification and which is a basic helix-loop-helix transcription factor transiently expressed in mesodermal populations, FOG2 (Friend of GATA4), which is associated physiologically with N-terminal zinc finger of GATA4 and modulate its transcriptional activity and CAMTA (Calmodulin binding transcription activator 2) which is a co-activator associated with NKX2-5.

Other tissues of mesodermal derivation, like adipose tissue, contain mesenchymal stem cells in the stromal vascular fraction (SVF) that could be differentiated in vitro to cardiomiocyte precursors. The SVF is composed of at least eight different cell populations as we already showed [9,10] and among other, endothelial cells, smooth muscle cells, and stem cells. The latest have been referred to as adipose tissue-derived stromal cells (ASCs). These reports have shown the ability for ASCs to undergo adipogenesis, chondrogenesis and osteogenesis.

Different approaches have been used to induce the differentiation of ASCs toward the cardiac lineage such as methylcellulose media [1 1 ], culturing with nuclear and cytoplasmic extracts from rat cardiomyocytes [12], co-culturing with neonatal rat cardiomyocytes [13] and use of the chemical agent 5-azacytidine [13,14]. Because of the use of products of animal origin or chemical agents that may affect genomic integrity and stability, none of the above-cited methods is compatible with the use of ASCs in the clinical practice. As already shown by Noseda et al.

[16] there are many biochemical pathways involved in cardiac muscle formation during embryogenesis and stem cell differentiation. Growth factors and cytokines acting on the main cardiac differentiation pathways have already been identified and could in theory supplement a defined culture medium to obtain functional cardiomyocytes physiologically indistinguishable from cardiomyocytes derived from cardiac tissue. In many developmental systems, Nodal, Activin, TGF , bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) promote cardiogenesis [17]. While non-canonical Wnts, which are β-catenin independent, have been shown to promote cardiac specification, canonical Wnts, which signal through β-catenin, were believed to function largely as inhibitor of cardiogenesis [18]. Multiple signalling pathways integrate and cross-regulate during cardiac differentiation. By specifically activating and blocking their activity, Activin, Nodal and Wnt were shown to be essential for cardiac specification of mesoderm, whereas bone morphogenetic (BMPs) proteins promote mesoderm formation through a Wnt/Activin/Nodal-dependent mechanism [19, 20]. Since the number growth factors, cytokines and other factors potentially involved in cardiogenesis is very high, it remains quite difficult to select and compose them in view of obtaining media for cardiogenic differentiation having with a significant activity.

SUMMARY

Studies on cardiogenesis induction are generally based on assessing the expression of one or more genes and cardiac trascription factors in the cells to be analysed: in particular, the expression of one such genes / factors in the treated cells is regarded as a marker of cardiogenesis. The present Applicant has now found that this approach is partially defective when used to study cardiogenesis in ASCs; in particular, a number of cardiac transcription factors which deemed typical for cardiogenesis, were found unsuitable or low-effective as markers for cardiogenesis from ASCs, such that any screening protocol to select suitable cardiogenesis-inducing media for ASCs based on these factors would fail to identify highly effective media. It was now found in particular that ACTB, GATA4, TBX5, BAF60C, TbxT8, CX43, commonly considered "cardiac" genes/transcription factors, are not effective markers of cardiogenesis from ASCs. Based on this finding, the Applicant has now selected a pool of other genetic markers for cardiogenesis (herein referred as "enhanced markers"), whose expression was found to be highly indicative of cardiogenic differentiation of ASCs: based on the upregulation of these genes, it was possible to select specific culture media for ASCs with enhanced cardiogenic differentiating potency, i.e. promoting cardiogenic differentiation with high specificity and high activity.

DESCRIPTION OF THE FIGURES

Figure 1 : Gene up-regulation tested by reverse transcription PCR of culture medium composed of cocktail no.14 after 21 days of culture of ASCs. Beta-actin represent the positive control and appears in all three lanes, non-induced (N.I.), induced (I) and heart biopsy (H). NKX2-5, MEF2C, HAND2, FOG and CAMTA are up-regulated only in induced cells and in positive control tissue heart biopsy.

Figure 2. Morphological changes after cardiac induction. From left: 4x magnified picture of non induced control ASCs, 4x picture of induced MSCs and 20x picture of induced ASCs.

Figure 3. Morphological changes after cardiac induction. Left: non induced control ASCs are bigger and with autosomes. Right: induced ASCs are smaller and without autosomes.

Figure 4. TEM morphological differences between non-induced and induced ASCs after cardiac induction. Left: non induced control cells. Right: induced ASCs with intracellular unorganized structures.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of a pool of enhanced genetic markers for cardiogenesis in ASCs; these markers have been used in a method to screen and select growth media which are highly active in promoting the cardiogenic differentiation of ASCs.

Said pool of enhanced genetic markers, representing an object of the present invention, consists in one or more genes chosen among NKX2-5, MEF2C, HAND2, HAND1 , CAMTA and FOG genes; the full characterization/description of these genes is available from standard literature (cf. e.g. www.genenames.org). Preferably, the pool of enhanced genetic markers includes at least three or at least four of said transcription factors; most preferably it includes them all.

In accordance with the invention, these genes are highly expressed in ASCs undergoing or having undergone cardiomiocyte differentiation. Based on this finding, a first object of the invention is a method to assess cardiogenic differentiation in ASCs. This method is used herein to select growth media with enhanced capability to stimulate cardiocyte differentiation of ASCs.

According to this method, the candidate growth media is incubated with the ASCs and, after a suitable incubation time, the expression of said genes is assessed. Candidate growth media usable in the present method are suitably chosen among those containing growth factors, interleukins, albumin, thrombin, insulin, and further proteins, peptides, aminoacids, vitamins, minerals, generally known as useful for growing stem cells; the candidate growth media further includes a suitable buffer, e.g. JMEM (Joklik's Minimum Essential Medium).

After a suitable incubation time with the ASCs, the harvested ASCs are challenged for up-regulation of one ore more among the present genes: NKX2-5, MEF2C, HAND2, HAND1 , CAMTA and FOG. Up-regulation of such genes is measured after induction of cells for a defined period of time, extracting mRNA and doing a RT-PCT to quantify the amount of the mRNAs of the specified genes; in the present invention the upregulation was evaluated by eye on an agarose gel.

The tested candidate growth media are then ranked according to the level of expression (up-regulation) of the following genes: NKX2-5, MEF2C, HAND2, HAND1 , CAMTA and FOG; the media showing the highest level of gene expression are considered those with the highest cardiomyocite-differentiating potency. The sensitivity/precision of the method is function of the number of genes considered (from one to five), with the highest precision reached when four or five of them are considered.

Another object of the invention is thus a growth medium selected by the present method. Further object is a growth medium with elevated cardiomyocyte- differentiating potency of ASCs, characterized by up-regulating/enhancing expression in said ASCs of one or more genes chosen among NKX2-5, MEF2C, HAND2, HAND1 , CAMTA and FOG. The potency of the medium is function of the number of the mentioned genes (from one to five), with the highest potency reached when the expression of four or five of them is enhanced.

Specific growth media with enhanced cardiogenic differentiating potency selected with the present method are those containing at least 12, preferably at least 15, most preferably all of the following components: ascorbic acid or salt thereof, insulin, transferrin, selenium, bFGF, BMP4, IGF1 , Activin A, TGF b1 , hThrombin, Retinoic acid, TNF a, VEGF, FGF4, FGF8, G-CSF, PDGF AB, PDGF BB, IL6, Cardiogenol, Cardiothrophin. Among them:

- the cited growth factors (GF), BMP4, G-CSF, cardiothropin, activin A, IL6, TNF and selenium are present in the medium preferably at concentrations comprised between 0.1 and 40 ng/mL;

- transferrin, insulin are present at concentrations between 0.05 and 10 g/mL

- retinoic acid and cardiogenol C are present at concentration between 1 and 20 nM.

- ascorbic acid or salt thereof is present at concentration between 10 and 100 mM.

- H thrombin is present at concentration between 0.1 and 10 U/mL

All the above concentrations are referred to the final growth medium.

The final growth medium is obtained by adding the above components to a standard basis medium for stem cells, including ingredients such a buffer, nutrients, either of serum origin or serum-free; typical examples of these ingredients are a JEM buffer, a HAM's F12 buffer, Primocin, Glutamine, Albumin, horse serum where desired, and mixtures thereof.

In particular, when a serum-free medium is desired, the serum ingredients are not used and, preferably, human albumin is added, in particular at concentrations between 1 and 10 % by weight, for example 5%.

Typical commercial suppliers for the above mentioned components and ingredients are listed in the Annex 1 .

The above defined medium is used for incubating the ACS at 37°C for a suitable time, tipically 10 days, this is followed by a further incubation time for another 10- 15 days, e.g. 1 1 days; this further incubation can be prosecuted in the same medium or, alternatively in a lighter medium lacking some of the above components; in particular, the lighter medium may be without one or more among BMP4, Activin A, TGF b1 , Rethinoic acid, TNFa, FGF 4/8/10, PDGF AB/BB, preferably lacking them all. The above mentioned extended incubation time is useful to obtain an extended differentiation of the cadiomyiocytes, so as to obtain, beyond the specific genetic expression, also the relevant morphological changes visible at the microscope. EXPERIMENTALS

The results presented here show the differentiation of adult human adipose tissue-derived multipotent stromal cells (ASCs) into non-beating precursors of cardiomyocytes, in a defined xeno- and serum-free culture induction medium supplemented with recombinant human growth factors upgradable to GMP conditions.

The reported experimental activity includes: (1 ) SVF isolation and phenotypic characterization; (2) Culture medium selection and cell culture conditions; (3) Gene expression. (RNA isolation, real-time PCR, detection); (4) Morphological analyses (TEM, immunofluorescence, etc.).

1. Isolation and characterization of Stromal Vascular Fraction (SVF).

The isolation protocol was performed according to the method described in the paper Zuk et al. [21 ], but using a 100ml syringe (Omnifix 100ml with Luer Adaptor, B. Braun AG, Melsungen, Germany) as a separation funnel (Patent pending). The protocol is based on the fact that adipose tissue and hydrophilic fluids spontaneously separate in two phases without need of centrifugation. The piston of the syringe is used to take in or to expel the solutions used to wash the sample, to dissociate the suctioned fat, or to extract the cells from the dissociated adipose tissue. The syringe is hold in a vertical position using a laboratory support stand with support rings.

2. Selection of cardiac transcription factors as markers of differentiation.

Total RNA was extracted from ASCs cultivated in serum defined medium, and positive control human heart RNA was obtained and isolated from heart biopsy. First-strand cDNA was synthesized with total RNA using the reverse transcriptase. cDNA samples were subjected to PCR amplification using primer pairs specific for cardiac-specific genes and transcription factors. Cells from heart biopsy showed the expected gene expression for the selected transcription factors (Table 1 ). Unexpectedly, ASCs also showed gene expression for the half of selected cardiac genes. These genes were considered unsuitable markers of cardiogenesis in ASCs and were discarded from consideration; a pool of enhanced markers was then selected from those genes having low or no expression in the native ASCs. In particular, based on Table 1 , the following transcription factors were selected as markers to monitor the induction of ASCs to cardiomyocyte precursors: NKX2- 5, MEF2C, HAND2, CAMTA and FOG.

Tab. 1. Expression of "cardiac" genes in biopsies and ASCs. x: Detectable gene expression. Recent work demonstrated that combination of the cardiogenic transcription factors GATA4, TBX5 and BAF60C can reprogram and drive cardiogenesis and that GATA4 and TBX5 are part of the combination that mediate the transition from mesoderm to cardiomyocytes. These results demonstrate that ASCs could have the potential to differentiate into cardiac precursors. 3. Cardiogenic Differentiation cocktails.

28 growth factors were assembled in 16 different cocktails. After expansion of ASCs in vitro, the cells were seeded into 6-wells plates and assayed during three weeks of culture with the 16 growth factor cocktails for their ability to induce cardiac gene expression. In particular, the ability to induce expression of the following genes has been investigated: NKX2-5, MEF2C, HAND2, CAMTA and FOG.

Table 2 summarizes all the tested cocktails. In this screening phase, the presence of differentiated cells was monitored by standard and quantitative RT-PCR for selected cardio-specific genes. The most informative ones are those not expressed in non-induced ASCs (see Table 1 ).

Tab. 2. Test of 16 cocktails on 21 days cultured ASCs. Cocktail number 14 has been chosen on the basis of the up-regulated gene's panel (X = present).

Cocktail 14 has been selected because of its most effective capacity in inducing cardiogenic phenotype, as reported by the up-regulation of the following cardiospecific genes: NKX2-5, MEF2C, HAND2, CAMTA and FOG. The obtained up-regulation is shown in Figure 1 .

Then the same cocktail no.14 has been tested with and without serum components. Indeed, we were interested in having a serum-free, xeno-free culture medium and we then produced the same no.14 cocktail with the addition of 5% human albumin only. The test has been repeated on 21 days cultured ASCs for the up-regulation of the same gene's panel and results evidenced that no.14 with human albumin is able to produce the same induction also without 1 % Horse serum addition (Table 3).

Tab. 3. Test of cocktail no.14 with 1% Horse Serum (14b and 14c) or 5% Human Albumin (14 SF) on 21 days cultured ASCs. Cocktail number 14 SF was able to induce cardiogenic gene's panel as the other cocktails added with 1% Horse Serum (X = present). Culture medium no 14 SF was then challenged for the up-regulation of the following cardiogenic genes: NKX2-5, MEF2C, HAND2, FOG and CAMTA. Results showed the same behaviour observed in Figure 1 for Horse Serum supplemented media, namely the up-regulation of all tested genes in control biopsies and in induced cells whereas no up-regulation was observed in non- induced cells.

We were then able to complete the Table 1 with a new column where inductions of cardiogenic genes are evidenced in induced cells (Table 4). These data demonstrate that our cardiogenic induction growth medium was able to induce the expression of NKX2-5, MEF2C, CAMTA, HAND2 and FOG genes as compared to the ASCs where these transcription factors were not induced.

Tab. 4. Expression of "cardiac" genes in biopsies in ASCs and Induced ASCs cells: X: detectable gene expression, A: no detectable gene expression. Clear boxes have not been tested. 4. Morphological tests.

Induced ASCs in culture showed morphological changes after 3 weeks of differentiation with our cardiogenic cocktail (Figure 2). Results are highly reproducible, as found after studying cells obtained from 10 patients and representative pictures are shown in Figure 2. Upon induction of the cardiogenic phenotype, ASCs show a change in the morphology being more round-shaped in the non-induced state and fusiform in the induced one.

Semi-thin sections stained by crystal violet and Fuchsin, when inspected under light microscopy, show big cells and presence of autosomes in non-induced cells, whereas induced cells appear smaller and withless autosomes as seen in Figure 3.

Further analysis by transmission electron microscopy (TEM) revealed the presence of unorganized tubular structures in the cell cytoplasm, as shown in Figure 4. In a further test, specific immunofluorescence staining of induced and non- induced cells was performed for NKX2-5, the master regulator of cardiac differentiation; the nuclear transcription factor NKX2-5 was evidenced by fluorescent signal in the nucleus, where it is expected to be found. Induction of further downstream phenotypes (actin, cardiac alpha-actinin, myosin light chain) was also studied. The induction of actin was evident by red staining of induced cells, whereas alpha-actinin, more specific for cardiogenic differentiation was also markedly increased in induced cells compared to non-induced cells where a nonspecific staining was visible around the DAPI-stained nuclei. Myosin, another muscle-specific marker for myo-differentiation, has been seen in a small part of the induced cells, whereas it could not be seen in any other non-induced cells.

Growth media preparation.

Two distinct culture media have to be prepared, one used for the first 10 days of culture and the second for the next 1 1 days. All reagents are prepared following Annex 1 and used at the specified concentrations in the Tables included. All growth factors and reagents are prepared in stock solutions and stored at various conditions. For every growth factor or reagent we specify the volume of stock solution to be used for 50 ml of culture medium.

Annex 1

Day 1 to day 10- Medium Composition

Day 10 to 21 - Medium Composition

In conclusion, the results discussed above show the selection of good cardiomyopoietic cocktails based on well-defined growth factors and cytokines. Furthermore we characterized the best time of application. All the analysis at the molecular level has consistently revealed an up-regulation of cardiac-specific transcription factors and in particular the expression of NKX2-5, MEF2C, CAMTA, HAND2 and FOG, which are not normally expressed by ASCs. Bibliography

[1 ] T. Thorn, N. Haase, W. Rosamond, V. J. Howard, J. Rumsfeld, T. Manolio, Z.-J. Zheng, K. Regal, C. O'Donnell, S. Kittner, D. Lloyd-Jones, D. C. Goff Jr, Y. Hong, R. Adams, G. Friday, K. Furie, P. Gorelick, B. Kissela, J. Marler, J. Meigs, V. Roger, S. Sidney, P. Sorlie, J. Steinberger, S. Wasserthiel-Smoller, M. Wilson, P. Wolf, and American Heart Association Statistics Committee and Stroke

Statistics Subcommittee, "Heart disease and stroke statistics-2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee," Circulation, vol. 1 13, no. 6, pp. e85-151 , Feb. 2006. [2] N. J. Palpant and J. M. Metzger, "Aesthetic cardiology: adipose-derived stem cells for myocardial repair," Curr Stem Cell Res Ther, vol. 5, no. 2, pp. 145- 152, Jun. 2010.

[3] Y. Zhu, T. Liu, K. Song, R. Ning, X. Ma, and Z. Cui, "ADSCs differentiated into cardiomyocytes in cardiac microenvironment," Mol. Cell. Biochem., vol. 324, no. 1-2, pp. 1 17-129, Apr. 2009.

[4] T. Brand, "Heart development: molecular insights into cardiac specification and early morphogenesis," Dev. Biol., vol. 258, no. 1 , pp. 1-19, Jun. 2003.

[5] H. Bartlett, G. J. C. Veenstra, and D. L. Weeks, "Examining the cardiac NK- 2 genes in early heart development," Pediatr Cardiol, vol. 31 , no. 3, pp. 335-341 , Apr. 2010.

[6] L. Zhao, D. Ju, Q. Gao, X. Zheng, and G. Yang, "Over-expression of NKX2- 5 and/or cardiac a-actin inhibit the contraction ability of ADSCs-derived

cardiomyocytes," Mol. Biol. Rep., vol. 39, no. 3, pp. 2585-2595, Mar. 2012.

[7] A. Armihan, C. Gandia, J. M. Garcia-Verdugo, E. Lledo, J. L. Mullor, J. A. Montero, and P. Sepulveda, "Cardiac transcription factors driven lineage- specification of adult stem cells," J Cardiovasc TransI Res, vol. 3, no. 1 , pp. 61-65, Feb. 2010.

[8] A. Bondue, G. Lapouge, C. Paulissen, C. Semeraro, M. lacovino, M. Kyba, and C. Blanpain, "Mespl acts as a master regulator of multipotent cardiovascular progenitor specification," Cell Stem Cell, vol. 3, no. 1 , pp. 69-84, Jul. 2008. [9] T. Tallone, C. Realini, A. Bohmler, C. Kornfeld, G. Vassalli, T. Moccetti, S. Bardelli, and G. Soldati, "Adult human adipose tissue contains several types of multipotent cells," J Cardiovasc Transl Res, vol. 4, no. 2, pp. 200-210, Apr. 201 1 .

[10] G. Astori, F. Vignati, S. Bardelli, M. Tubio, M. Gola, V. Albertini, F. Bambi, G. Scali, D. Castelli, V. Rasini, G. Soldati, and T. Moccetti, '"In vitro' and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells," J Transl Med, vol. 5, p. 55, 2007.

[1 1 ] A. Sliwa, A. Balwierz, B. Kiec-Wilk, A. Polus, A. Knapp, and A. Dembinska- Kiec, "Differentiation of human adipose tissue SVF cells into cardiomyocytes," Genes Nutr, vol. 4, no. 3, pp. 195-198, Sep. 2009.

[12] K. G. Gaustad, A. C. Boquest, B. E. Anderson, A. M. Gerdes, and P. Collas, "Differentiation of human adipose tissue stem cells using extracts of rat

cardiomyocytes," Biochem. Biophys. Res. Commun., vol. 314, no. 2, pp. 420-427, Feb. 2004.

[13] R. Madonna, J. T. Willerson, and Y.-J. Geng, "Myocardin a enhances telomerase activities in adipose tissue mesenchymal cells and embryonic stem cells undergoing cardiovascular myogenic differentiation," Stem Cells, vol. 26, no. 1 , pp. 202-21 1 , Jan. 2008. [14] N. J. Palpant, S. Yasuda, O. MacDougald, and J. M. Metzger, "Non- canonical Wnt signaling enhances differentiation of Sca1 +/c-kit+ adipose-derived murine stromal vascular cells into spontaneously beating cardiac myocytes," J. Mol. Cell. Cardiol., vol. 43, no. 3, pp. 362-370, Sep. 2007.

[15] A. van Dijk, H. W. M. Niessen, B. Zandieh Doulabi, F. C. Visser, and F. J. van Milligen, "Differentiation of human adipose-derived stem cells towards cardiomyocytes is facilitated by laminin," Cell Tissue Res., vol. 334, no. 3, pp. 457-467, Dec. 2008.

[16] M. Noseda, T. Peterkin, F. C. Simoes, R. Patient, and M. D. Schneider, "Cardiopoietic factors: extracellular signals for cardiac lineage commitment," Circ. Res., vol. 108, no. 1 , pp. 129-152, Jan. 201 1 .

[17] A. Behfar, R. S. Faustino, D. K. Arrell, P. P. Dzeja, C. Perez-Terzic, and A. Terzic, "Guided stem cell cardiopoiesis: discovery and translation," J. Mol. Cell. Cardiol., vol. 45, no. 4, pp. 523-529, Oct. 2008.

[18] P. W. Burridge, G. Keller, J. D. Gold, and J. C. Wu, "Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct

reprogramming," Cell Stem Cell, vol. 10, no. 1 , pp. 16-28, Jan. 2012.

[19] J. M. Alexander and B. G. Bruneau, "Lessons for cardiac regeneration and repair through development," Trends Mol Med, vol. 16, no. 9, pp. 426-434, Sep. 2010.

[20] E. Willems, M. Lanier, E. Forte, F. Lo, J. Cashman, and M. Mercola, "A chemical biology approach to myocardial regeneration," J Cardiovasc TransI Res, vol. 4, no. 3, pp. 340-350, Jun. 201 1 .

[21 ] P. A. Zuk, M. Zhu, P. Ashjian, D. A. De Ugarte, J. I. Huang, H. Mizuno, Z. C. Alfonso, J. K. Fraser, P. Benhaim, and M. H. Hedrick, "Human adipose tissue is a source of multipotent stem cells," Mol. Biol. Cell, vol. 13, no. 12, pp. 4279-4295, Dec. 2002.

Claims

1 . A method to asses cardiogenic differentiation of adipose tissue-derived stromal cells (ASCs), characterized by using, as genetic markers of said differentiation, one or more among NKX2-5, MEF2C, HAND2, HAND1 , CAMTA and FOG genes.

2. The method of claim 1 , where said markers are three or more among NKX2- 5, MEF2C, HAND2, HAND1 , CAMTA and FOG

3. The method of claims 1 -2, where the used markers are at least: NKX2-5, MEF2C, HAND2, CAMTA and FOG.

4. The method of claims 1 -3, for use in selecting a cardiogenic-differentiating medium for ASCs.

5. Cardiogenic-differentiating medium for ASCs, selected by the method of claims 1 -4.

6. Cardiogenic-differentiating medium for ASCs characterized by enhancing, in said ASCs, the expression of one or more among NKX2-5, MEF2C, HAND2, H AN D1 , CAMTA and FOG.

7. The medium of claim 6, characterized by enhancing, in said ASCs, the expression of three or more among NKX2-5, MEF2C, HAND2, HAND1 ,

CAMTA and FOG.

8. The medium of claims 6-7, characterized by enhancing, in said ASCs, the expression of at least: NKX2-5, MEF2C, HAND2, CAMTA and FOG.

9. The medium of claim 6-8, comprising at least 12 of the following components: ascorbic acid, ITS, bFGF, BMP4, IGF1 , Activin A, TGF b1 , H Thrombin, Retinoic acid, TNF a, VEGF, FGF4, FGF8, G-CSF, PDGF AB, PDGF BB, IL6, Cardiogenol, Cardiothrophin.

10. The medium of claim 6-9, comprising at least 15 of the following components: ascorbic acid, ITS, bFGF, BMP4, IGF1 , Activin A, TGF b1 ,

H Thrombin, Retinoic acid, TNF a, VEGF, FGF4, FGF8, G-CSF, PDGF AB, PDGF BB, IL6, Cardiogenol, Cardiothrophin.

1 1 . The medium of claims 6-10, comprising the following components: ascorbic acid, ITS, bFGF, BMP4, IGF1 , Activin A, TGF b1 , H Thrombin,

Retinoic acid, TNF a, VEGF, FGF4, FGF8, G-CSF, PDGF AB, PDGF BB, IL6, Cardiogenol, Cardiothrophin.

12. The medium of claims 6-1 1 , further supplemented with nutrients suitable for ASCs, preferably including: JEM, F12, Primocin, Glu.

13. The medium of claim 12, containing albumin and being serum-free.