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Decellularized Extracellular Matrices and Cardiac Differentiation: Study on Human Amniotic Fluid-Stem Cells

Cell therapy with a variety of stem populations is increasingly being investigated as a promising regenerative strategy for cardiovascular (CV) diseases. Their combination with adequate scaffolds represents an improved therapeutic approach. Recently, several biomaterials were investigated as scaffolds for CV tissue repair, with decellularized extracellular matrices (dECMs) arousing increasing interest for cardiac tissue engineering applications. The aim of this study was to analyze whether dECMs support the cardiac differentiation of CardiopoieticAF stem cells. These perinatal stem cells, which can be easily isolated without ethical or safety limitations, display a high cardiac differentiative potential. Differentiation was previously achieved by culturing them on Matrigel, but this 3D scaffold is not transplantable. The identification of a new transplantable scaffold able to support CardiopoieticAF stem cell cardiac differentiation is pivotal prior to encouraging translation of in vitro studies in animal model preclinical investigations. Our data demonstrated that decellularized extracellular matrices already used in cardiac surgery (the porcine CorTMPATCH and the equine MatrixPatchTM) can efficiently support the proliferation and cardiac differentiation of CardiopoieticAF stem cells and represent a useful cellular scaffold to be transplanted with stem cells in animal hosts.


Introduction
Cardiovascular (CV) diseases represent some of the main causes of mortality in worldwide [1], comprising 31% of global death [2][3][4]. The generation of fully differentiated cardiomyocytes (CMs) and In the first experimental condition, both the undifferentiated hiPSCs and the Cardiopoietic AF engrafted and proliferated easily on the dECMs and, when induced to differentiate, the viability was always comparable to the control cells differentiated on the Matrigel ® .
Although a slight foreseeable drop in cell numbers was observed when the cells were cultured under cardiac permissive induction conditions (24 h of BMP4 and ActA followed by 72h of VEGF stimulation for mesodermal and cardiac induction, respectively), the cell vitality remained high (>82 ± 5% in hiPSCs and >76 ± 4% in Cardiopoietic AF) after 15 days of differentiation in all experimental conditions.
In contrast, the second experimental condition did not give the same encouraging results; only a small percentage of hiPSC-and Cardiopoietic AF-derived CMs were able to attach to the dECMs once dissociated and no viable cells were present on the matrices after 72 h. Dissociated Cardiopoietic AFderived CMs were also unable to adhere and proliferate when reseeded on the Matrigel ® control ( Figure 2). These observations suggest that both hiPSCs the Cardiopoietic AF-derived CMs are deeply threatened by enzymatic dissociation, hampering their abilities to restore their cell-matrix and cellcell interactions, thereby affecting their viability. (2) the Cardiopoietic AF and the hiPSCs were differentiated in monolayer onto THE Matrigel ® for 12 days and then dissociated and moved to the Matrix Patch TM or Cor TM PATCH for 72 h.
In the first experimental condition, both the undifferentiated hiPSCs and the Cardiopoietic AF engrafted and proliferated easily on the dECMs and, when induced to differentiate, the viability was always comparable to the control cells differentiated on the Matrigel ® .
Although a slight foreseeable drop in cell numbers was observed when the cells were cultured under cardiac permissive induction conditions (24 h of BMP4 and ActA followed by 72h of VEGF stimulation for mesodermal and cardiac induction, respectively), the cell vitality remained high (>82 ± 5% in hiPSCs and >76 ± 4% in Cardiopoietic AF) after 15 days of differentiation in all experimental conditions. In contrast, the second experimental condition did not give the same encouraging results; only a small percentage of hiPSC-and Cardiopoietic AF-derived CMs were able to attach to the dECMs once dissociated and no viable cells were present on the matrices after 72 h. Dissociated Cardiopoietic AF-derived CMs were also unable to adhere and proliferate when reseeded on the Matrigel ® control (Figure 2). These observations suggest that both hiPSCs the Cardiopoietic AF-derived CMs are deeply threatened by enzymatic dissociation, hampering their abilities to restore their cell-matrix and cell-cell interactions, thereby affecting their viability. After 15 days of differentiation culture following the first experimental condition either on the dECMs or on the Matrigel ® internal control, we analyzed hiPSC-and Cardiopoietic AF-derived CM expression of cTnT, α-MHC, and α-SA, specific sarcomeric proteins recognized as "late" cardiac markers. Sarcomeric proteins represent the structural building blocks of heart muscle, which are essential for contraction and relaxation [26]. Interestingly, differentiated cells obtained from both hiPSCs and Cardiopoietic AF expressed high levels of cTnT, α-MHC, and α-SA, comparable in all the experimental conditions ( Figure 3 and Table 1). In particular, the percentage and fluorescence intensity of the analyzed proteins were stackable for cTnT and a-MHC in hiPSCs and Cardiopoietic AF cells cultured in the dECMs and Matrigel ® , while α-SA of Cardiopoietic AF cultured in the Matrix Patch TM was less expressed, although still at a high percentage, than the same cells differentiated in Matrigel ® . α-SA of the hiPSCs and Cardiopoietic AF cells cultured in the Cor TM PATCH remaiedn comparable to those differentiated in Matrigel ® , although slightly less expressed (Table 1 and Figure 3). Nevertheless, immunofluorescence images displayed clearly that the sarcomeric striations typical of α-SA and cTnT localization were present in all culture conditions, indicating that both dECMs are permissive to cardiac differentiation (Table 1 and Figure 3). After 15 days of differentiation culture following the first experimental condition either on the dECMs or on the Matrigel ® internal control, we analyzed hiPSC-and Cardiopoietic AF-derived CM expression of cTnT, α-MHC, and α-SA, specific sarcomeric proteins recognized as "late" cardiac markers. Sarcomeric proteins represent the structural building blocks of heart muscle, which are essential for contraction and relaxation [26]. Interestingly, differentiated cells obtained from both hiPSCs and Cardiopoietic AF expressed high levels of cTnT, α-MHC, and α-SA, comparable in all the experimental conditions ( Figure 3 and Table 1). In particular, the percentage and fluorescence intensity of the analyzed proteins were stackable for cTnT and a-MHC in hiPSCs and Cardiopoietic AF cells cultured in the dECMs and Matrigel ® , while α-SA of Cardiopoietic AF cultured in the Matrix Patch TM was less expressed, although still at a high percentage, than the same cells differentiated in Matrigel ® . α-SA of the hiPSCs and Cardiopoietic AF cells cultured in the Cor TM PATCH remaiedn comparable to those differentiated in Matrigel ® , although slightly less expressed (Table 1 and Figure 3). Nevertheless, immunofluorescence images displayed clearly that the sarcomeric striations typical of α-SA and cTnT localization were present in all culture conditions, indicating that both dECMs are permissive to cardiac differentiation (Table 1 and Figure 3).
Flow cytometry and immunofluorescence revealed the dramatic induction of the L-type calcium channel CACNA1C and SERCA2 proteins, two calcium pumps essential for excitation-contraction coupling that reside in the sarcolemma and sarcoplasmic reticulum of CMs, respectively (Table 1 and Figure 4a). In the hiPSCs and Cardiopoietic AF cells differentiated in the dECMs and in Matrigel ® , CACNA1C was localized mainly in the perinuclear area and marked the cytoplasmic membrane in some cells, probably indicating a homing of the protein in the sarcolemma of more mature CMs. On the other hand, SERCA2 immunolabeling showed the typical reticulated organization of the sarcoplasmic reticulum ( Figure 4b). Flow cytometry and immunofluorescence revealed the dramatic induction of the L-type calcium channel CACNA1C and SERCA2 proteins, two calcium pumps essential for excitation-contraction coupling that reside in the sarcolemma and sarcoplasmic reticulum of CMs, respectively (Table 1 and Figure 4a). In the hiPSCs and Cardiopoietic AF cells differentiated in the dECMs and in Matrigel ® , CACNA1C was localized mainly in the perinuclear area and marked the cytoplasmic membrane in some cells, probably indicating a homing of the protein in the sarcolemma of more mature CMs. On the other hand, SERCA2 immunolabeling showed the typical reticulated organization of the sarcoplasmic reticulum (Figure 4b).    Finally, the beating colonies appeared from 8 days of culture and increased until 15 days on almost all of the substrates. hiPSC-and Cardiopoietic AF-derived CMs differentiated in both dECMs showed percentages of spontaneous beating foci comparable to or even higher than the same cells differentiated in Matrigel (Figure 5), demonstrating once again that both the Cor TM PATCH and Matrix Patch TM could be considered suitable scaffolds for the adhesion and the proliferation of stem cells from different sources and permissive for differentiation toward cardiac lineage. Finally, the beating colonies appeared from 8 days of culture and increased until 15 days on almost all of the substrates. hiPSC-and Cardiopoietic AF-derived CMs differentiated in both dECMs showed percentages of spontaneous beating foci comparable to or even higher than the same cells differentiated in Matrigel ( Figure 5), demonstrating once again that both the Cor TM PATCH and Matrix Patch TM could be considered suitable scaffolds for the adhesion and the proliferation of stem cells from different sources and permissive for differentiation toward cardiac lineage.

Discussion
The ideal goal of stem cell therapy is to substitute necrotic or dysfunctional cardiac tissue with new competent CMs derived from stem cells [27,28]. Currently, the only cells with proven promising results regarding cardiogenic potential are hiPSCs. The possibility to obtain functionally mature CMs from patient-specific hiPSCs is a big challenge for clinical use in tailored cell therapy [5,[29][30][31][32][33], with their epigenetic instability still considered an unsolved issue [10]. The recent discovery that Cardiopoietic AF could circumvent any ethical and safety concerns and provide effective cell replacement therapy for cardiac diseases, particularly in congenital heart defect repair applications, [2, 34,35] opened the door to a new scenario.
For the first time to our knowledge, Cardiopoietic AF (a new and safe source of stem cells) were demonstrated to be able to proliferate on dECMs and generate CMs with high efficiency in this work. In line with the literature [7,36] cardiac differentiation from hiPSCs and Cardiopoietic AF cells was observed to be optimal in terms of both efficacy and efficiency when cultured in a monolayer on Matrigel ® , a 3D matrix known to support cells in "in vitro" studies, but is unfortunately not transplantable for safety reasons [37]. In this study, we replaced the Matrigel ® with dECMs and investigated the biological response of Cardiopoietic AF cells to two engineered patches, the pericardial Cor TM PATCH and the Matrix Patch TM . Both technologies are decellularized membranes, one derived from porcine intestinal submucosa, the other from equine pericardium, which are commonly used in clinical practice, mainly as patches to restore valves and vascular tissue in need of repair; when resorbed, they allow the formation of nonfibrotic connective tissue. Neither were previously analyzed as possible transplantable scaffolds for CMs generated "in vitro". dECMs were widely explored as natural scaffolds for cardiac tissue engineering applications because they offer many unique advantages, such as preservation of organ-specific ECM microstructure and composition, retention of tissue-mimetic mechanical properties, and biochemical cues in favor of subsequent recellularization [12,38,39]. In this study, we exploited two dECMs from small intestinal submucosa and bovine-derived pericardium that were previously successfully tested in clinical studies [40] dECM scaffolds can be implanted during cardiac surgery intervention onto injured myocardial regions since they offer a structural support to the damaged myocardium and stimulate endogenous mechanisms of tissue repair, such as vasculogenesis [40]. Our data suggest their potential use as biological scaffolds to support Cardiopoietic AF cell proliferation and cardiac differentiation, with the ideal goal of implantation at the site of the infarction to restore tissue function. Interestingly, dECMs derived from both cardiac and intestinal tissues showed similar results, with no substantial differences in cellular adhesion or differentiation efficiency.
Another important observation was that both hiPSCs and Cardiopoietic AF needed to be cultured from the beginning on the dECMs (first experimental condition of this manuscript) in order to guarantee proper engrafting and to maintain their differentiation features. This was unsurprising, as previous studies showed that most of the murine embryonic stem cell (mESC)-derived CMs failed to attach to a variety of polymer substrates at the end of the differentiation process. It is therefore probable that CMs need a more complex environment than 2D or 3D scaffolds enriched in cardioprotective factors to attach and survive after dissociation [41]. In addition, Van Deel et al. showed that the commonly used enzymatic detaching methods, such as Trypsin, Detachin, and Accutase, are lethal to adult CMs, altering the integrity of the cells [42]. Thus, the failure of CMs obtained from hiPSCs and Cardiopoietic AF cells to attach onto dECMs was probably due to the fact that, after enzymatic digestion, the CMs were not able to recreate cell-cell and cell-scaffold connections. However, further studies are necessary to address this point.
Cardiopoietic AF stem cells attached to and proliferated easily on the two engineered scaffolds, and, if induced to differentiate, gave rise to CMs that showed typical sarcomere striation, expressed mature CMs hallmark proteins (α-AS, α-MHC, and cTnT) [5,43] and showed strong CACNA1 and SERCA2 expression, two L-type calcium channels essential for the so-called "Ca 2+ -induced Ca 2+ release", the mechanism crucial for excitation-contraction coupling in mammalian cardiac muscle. Cardiopoietic AF-derived CMs cultured on dECMs showed a proper homogeneous cardiac phenotype [43] and expressed cardiac-specific functional markers comparable to those obtained using the Matrigel ® culture. Finally, hiPSC-derived CMs differentiated using both dECMs showed percentages of spontaneous beating foci comparable to the same cells differentiated using Matrigel ® , while the percentage of the contracting area in Cardiopoietic AF-derived CMs was surprisingly significantly higher in both the Cor TM PATCH and Matrix Patch TM than in the control cells. Even if this percentage never exceeded 30% of the total area, it was still a promising result, since very few groups were able to obtain beating CMs from amniotic stem cells without prior reprogramming [2, 44,45]. This was possibly because, as expected [5,46,47] CMs with different levels of maturity were obtained, and a scaffold with a rigid elasticity niche may strongly promote beating induction, in line with what was reported by Hirata and colleagues [48].
In conclusion, these data confirm the efficacy of both the analyzed dECMs as cell carriers for ensuring phenotypic and functional Cardiopoietic AF-derived CMs, driving new challenges for producing biomimetic cardiac patches in tailored regenerative medicine.
The main limitation of this study was the lack of specific analysis of the calcium-handling capabilities of Cardiopoietic AF-derived CMs differentiated using the Cor TM PATCH and the Matrix Patch TM . More efforts are needed to characterize the electrophysiological properties and to promote of the homogeneity of Cardiopoietic AF-derived CMs prior to encouraging their transplantation in animal models.

Isolation and Culture of CardiopoieticAF and hiPSC Cells
AF samples were obtained from 8 women undergoing amniocentesis for prenatal diagnosis at 16-17 weeks of pregnancy after written informed consent, in accordance with the Declaration of Helsinki. All samples presented normal diploid male karyotypes, as evidenced by cytogenetic investigation. The mean (±SD) maternal age at amniocentesis was 37.9 ± 2.6 years. The study was approved by the local ethics committee and all experiments were performed in accordance with relevant guidelines and regulations.
Cardiopoietic AF cells were isolated as previously described on the basis of the phenotypic pattern

Culture in Monolayer and Cardiac Differentiation
Cardiopoietic AF and hiPSCs were seeded either onto Matrigel ® (Corning, Flintshire, UK)-coated plates or onto two dECMs ((Cor TM PATCH (CorMatrix, Roswell, GA, USA) or Matrix Patch TM (Auto Tissue Berlin GmbH)) using two methods: a) The cells were seeded onto the scaffold before starting the differentiation protocol and were kept on the dECMs for the duration of the process, followed by analysis; b) the cells were differentiated onto Matrigel ® and after 12 days of differentiation were detached using 0.025% Tryspin-EDTA (Invitrogen Carlsband, CA, USA) and distributed on each dECM or back to the Matrigel ® control. Sample were cultured in differentiation conditions for 72 h and subsequently analyzed. The cells were cultured onto the dECM as described above, then, when 80-90% confluence was reached, the medium was changed to RPMI 1640 (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with B-27 minus insulin (GIBCO, Thermo Fischer Scientific, Waltham, MA, USA), 50 µg/mL ascorbic acid (Sigma-Aldrich, Saint Louis, MO, USA) and 10 µM 5-aza-2 -deoxycytidine (5-Aza), and differentiation was induced by exposure for 24 h to activin A (50 ng/mL, R&D Systems, Minneapolis, MN, USA) and BMP4 (25 ng/mL, R&D Systems, Minneapolis, MN, USA), followed by treatment for 72 h with VEGF (10 ng/mL, R&D Systems, Minneapolis, MN, USA). Cells were analyzed after 15 days of differentiation. Beating foci were observed and quantified microscopically as the percentage of the beating area inside the whole plate [50,51].
The values obtained in the absence of cells were considered to be background and subtracted from the optical density values of the samples. Five independent experiments were performed under the same experimental conditions.
Cytometric analysis was performed with a Cytoflex cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) or CytExpert Acquisition and Analysis Software (Beckman Coulter, Brea, CA, USA).

Statistical Analysis
All quantitative data were presented as the mean ± SD. Statistical comparisons were performed using one-way analysis of variance (ANOVA) and Student's t-test [55]. The level of significance was set at p < 0.05.